UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS 


COLLEGE  OF  AGRICULTURE 

AGRICULTURAL  EXPERIMENT  STATION 

BERKELEY.  CALIFORNIA 


ENOLOGICAL  INVESTIGATIONS 


BY 


FREDERIC  T.  BIOLETTI  and  WILLIAM  V.  CRUESS 


BULLETIN  No.  230 

(BERKELEY,  CALIFORNIA,  AUGUST,  1912) 


Friend  Wm.  Richardson,  Superintendent  of  State  Printing 

sacramento,  california 

1912 


Benjamin  Ide  Wheeler,  President  of  the  University. 

EXPERIMENT  STATION  STAFF. 

E.  J.  Wickson,  M.A.,  Director  and  Horticulturist. 

E.  W.  Hilgard,  Ph.D.,  LL.D.,  Chemist   (Emeritus). 
W.  A.  Setchell,  Ph.D.,  Botanist. 

Leroy  Anderson,  Ph.D.,  Dairy  Industry. 
M.  E.  Jaffa,  M.S.,  Nutrition  Expert. 

R.  H.  Loughridge,  Ph.D.,  Soil  Chemist  and  Physicist  (Emeritus). 
C.  W.  Woodworth,  M.S.,  Entomologist. 

Ralph  E.   Smith,  B.S.,   Plant  Pathologist  and  Superintendent  of  Southern  California 
Pathological  Laboratory  and  Experiment  Station. 

F.  R.  Marshall,  B.S.A.,   Animal  Industry. 

G.  W.  Shaw,  M.A.,  Ph.D.,  Experimental  Agronomist  and  Agricultural  Technologist,  in 

charge  of  Cereal  Stations. 

B.  A.  Etcheverry,  B.S.,  Irrigation  Expert. 
F.  T.  Bioletti,  M.S.,  Viticulturist. 

W.  T.  Clarke,  B.S.,  Assistant  Horticulturist  and  Superintendent  of  University  Exten- 
sion in  Agriculture. 
John  S.  Burd,  B.S.,  Chemist,  in  charge  of  Fertilizer  Control. 
J.  E.  Coit,  Ph.D.,  Pomologist,  Riverside. 

C.  B.  Lipman,  Ph.D.,  Soil  Chemist  and  Bacteriologist. 

George    E.    Colby,    M.S.,    Chemist    (Fruits,    Waters,    and    Insecticides),    in    charge    of 

Chemical  Laboratory. 
H.  J.  Quayle,  M.S.,  Assistant  Entomologist. 
H.  M.  Hall,  Ph.D.,  Assistant  Botanist. 
C.  M.  Haring,  D.V.M.,  Veterinarian  and  Bacteriologist. 
E.   B.   Babcock,  B.S.,  Agricultural  Education. 
W.  B.  Herms,  M.A.,  Assistant  Entomologist. 
W.  T.  Horne,  B.S.,  Assistant  Plant  Pathologist. 
L.  M.  Davis,  B.S.,  Assistant  Dairy  Industry. 
W.  W.  Bonns,  M.S.,  Assistant  Pomologist. 

A.  J.  Gaumnitz,  M.S.,  Assistant  Agronomist,  University  Farm,  Davis. 
T.  F.  Hunt,  B.S.,  Assistant  Plant  Pathologist. 

P.  L.  McCreary,  B.S.,  Chemist  in  Fertilizer  Control. 

E.  H.  Hagemann,  Assistant  in  Dairying,  Davis. 

R.  M.  Roberts,  Farm  Manager,  University  Farm,  Davis. 
J.  I.  Thompson,  B.S.,  Assistant  Animal  Industry,  Davis. 
J.  C.  Bridwell,  B.S.,  Assistant  Entomologist. 
L.  Bonnet,  I.A.,  Assistant  Viticulturist. 

F.  C.  H.  Flossfeder,  Assistant  in  Viticulture,  University  Farm,  Davis. 
P.  L.  Hibbard,  B.S.,  Assistant  Fertilizer  Control  Laboratory. 

C.  H.  McCharles,  M.S.,  Assistant,  Agricultural  Chemical  Laboratory. 

B.  A.  Madson,  B.S.A.,   Assistant  Experimental  Agronomist. 

Walter  E.  Packard,  M.S.,  Field  Assistant  Imperial  Valley  Investigation,   El  Centro. 
S.  S.  Rogers,  B.S.,  Assistant  Plant  Pathologist,  Plant  Disease  Laboratory,  Whittier. 

C.  O.  Smith,  M.S.,  Assistant  Plant  Pathologist,  Plant  Disease  Laboratory,  Whittier. 

E.  H.   Smith,  M.S.,  Assistant  Plant  Pathologist. 

C.  L.  Roadhouse,  D.V.M.,  Assistant  in  Veterinary  Science. 

F.  M.  Hayes,  D.V.M.,  Assistant  Veterinarian. 
P.  S.  Burgess,  M.S.,  Assistant  Soil  Chemist. 
W.  F.  Gericke,  B.S.,  Assistant  Soil  Chemist. 

M.  E.  Stover,  B.S.,  Assistant  in  Agricultural  Chemical  Laboratory. 
W.  H.  Volck,  Field  Assistant  in  Entomology,  Watsonville. 
E.  L.  Morris,  Field  Assistant  in  Entomology,  San  Jose. 

E.  E.  Thomas,  B.S.,  Assistant  Chemist,  Plant  Disease  Laboratory,  Whittier. 
A.  B.  Shaw,  B.S.,  Assistant  in  Entomology. 

G.  P.  Gray,  M.S.,  Chemist  in  Insecticides. 

H.   D.   Young,  B.S.,   Assistant   in  Agricultural   Chemistry,   Plant   Disease   Laboratory, 

Whittier. 
A.  R.  Tyler,  B.S.,  Assistant  in  Plant  Pathology,  Plant  Disease  Laboratory,  Whittier 
W.  V.  Cruess,  B.S.,  Assistant  in  Zymology. 
J.  F.  Mitchell,  D.V.M.,  Assistant  in  Veterinary  Laboratory. 
W.  A.  Boys,  B.S.,  Assistant  Cerealist. 
M.  R.  Miller,  B.S.,  Assistant  Chemist  in  Insecticides. 

F.  H.  Wilson,  B.S.,  Assistant  in  Soil  Chemistry. 
W.  M.  Mertz,  Assistant  in  Pomology,   Riverside. 

Anna  M.  Lute,  A.B.,  Scientific  Assistant  U.  S.  Department  of  Agriculture. 
J.  C.  Roper,  Patron,  University  Forestry  Station,  Chico. 
E.  C.  Miller,  Foreman,  Forestry  Station,  Chico. 

D.  L.   Bunnell,  Secretary  to  Director. 


CONTENTS. 

Page. 
I.     INTRODUCTION 23 

II.     SULFUROUS  ACID  IN  WINE-MAKING 25 

A.  General  Considerations 25 

(a)      Historical   25 

(&)      Modern  Developments 25 

(c)      Opinions  of  Experts 25 

{d)      Legal  Limitations 2(> 

B.  Properties,  Preparation,  Use  and  Effects 2T 

(a)      Properties:     1,  Physical ;    2,  Chemical 27 

(&)     Preparation     and     Use     in     Wineries:     1,     Sulfur     fumes; 

2,  Liquid;    3,  Solutions;    4,  Solid  forms 28 

(c)  Transformation  of  Sulfurous  Acid:     1,  Disappearance  from 

various    solutions ;    2,    Disappearance    from    grape    must ; 

3,  Winery  tests  ;  4,  Disappearance  from  wine  ;  5,  Final  forms       36 

(d)  Influence  of  Composition  and  Treatment  of  Must  on  Fermen- 

tation inhibiting  powers  of  S02 :     1,  Acidity ;    2,  Sugar ; 

3,  Limpidity  ;    4,  Heating  ;    5,  Ripeness  of  Grapes 45 

(e)  Effects  on  Micro-organisms:     1,  Comparison  of  free  and  com- 

bined forms ;  2,  Effect  on  pure  cultures ;  3,  Effect  on  mix- 
tures of  organisms  in  must ;  4,  Effects  in  defecation ; 
5,  Changes  in  susceptibility  of  wine  yeast 49 

III.     UTILITY  AND  METHODS  OF  APPLICATION  OF  PURE  YEAST 

IN  WINE-MAKING G4 

(a)      Present  Status  of  Pure  Yeast  in  Wine-Making 64 

(6)      Micro-organisms    of    Californian    Grape:      1,    Grapes    from 

Davis  ;    2,  from  Contra  Costa  County  ;    3,  from  Acampo 65 

(c)  Methods  of  Transmitting  Pure  Yeast 69 

(d)  Methods     of     Use     in     Wineries:     1,     Direct     applications; 

2,  Preparation  of  a  starter 72 

(e)  Rejuvenation  and  Increase  of  the  Pure  Cultures 72 

(f)  Directions  for  Propagating  Yeast  in  the  Winery:     1,   Solid 

Cultures;    2,  Liquid  Cultures 74 

(g)  Use  of  Starter 76 

(h)      Stage  of  Maximum  Efficiency  of  a  Starter 77 

(i)      Simple  Yeast  Apparatus  for  Wineries 82 

(;')      Proper  Working  of  the  Yeast  Propagator 85 

IV.  WINERY  TESTS  OF  SULFUROUS  ACID  AND  PURE  YEAST 86 

(a)      Objects  and  Nature  of  the  Tests 86 

(6)      Changes  in  the  Kind  or  Number  of  the  Micro-organisms 86 

(c)  Duration  and  Course  of  the  Fermentation:     1,  Fermentation 

curves  ;  2,  Red  wine  fermentations  ;  3,  White  wine  fermen- 
tations           88 

(d)  Quality    of    the    Wines:     1,     Sugar;    2,     Volatile    acidity; 

3,  Fixed  acidity  ;  4,  Alcohol ;  5,  Color ;  6,  Condition ;  7,  Sul- 
furous Acid  remaining  in  Wine 107 

V.     SUMMARY  AND  CONCLUSIONS 11G 


LIST  OF  TABLES. 

Page. 

1.  S02  Produced  by  burning  Sulfur  in  a  Closed  Space 32 

2.  Absorption  of  S02  in  a  barrel  after  Maximum  Sulfuring 34 

3.  Disappearance  of  S02  from  Various  Solutions 36 

4.  Disappearance  of  free  S02  from  Various  Musts 38 

5.  Free  S02  remaining  in  Must  of  Raisins  and  of  Fresh  Grapes 39 

6.  Disappearance  of  Total  and  Free  S02  from  Fermenting  Red  Wine 40 

7.  Disappearance  of  S02  from  Wine  after  Fermentation 41 

8.  Increase  of  Sulfates  due  to  Use  of  S02  (Laboratory  Tests) 43 

9.  Increase  of  Sulfates  due  to  Use  of  S02  (Winery  Tests) 44 

10.  Loss  of  Sulfates  in  Wine  after  Fermentation 44 

11.  Influence  of  Acidity  of  Must  on  Effect  of  S02 45 

12.  Influence  of  Solid  Matter  of  the  Grape  on  Effect  of  S02 46 

13.  Influence  of  the  Heating  of  Must  on  the  Effect  of  S02 47 

14.  Influence  of  Degree  of  Ripeness  of  Grapes  on  Effect  of  S02 48 

15.  Comparison  of  Effects  of  Free  with  those  of  Combined  S02 49 

16.  Comparison  of  Effects  of  Free  with  those  of  Combined  S02 ■    50 

17.  Amount  of  Free  S02  which  will  permit  Fermentation  to  Start 51 

18.  Effect  of  S02  on  Multiplication  of  Organisms 52 

19.  Modification  of  Relative  Numbers  of  Micro-organisms  in  Natural  Must 53 

20.  Elimination  of  Micro-organisms  by  Defecation 55 

21.  Elimination  of  Micro-organisms  in  1,500  gallon  Defecating  Vat 56 

22.  Elimination  of  Micro-organisms  in  Small  Vat 57 

23.  Exposure  of  Yeast  to  increasing  Doses  of  S02 58 

24.  Comparison  of  Accustomed  with  Unaccustomed  Yeast 59 

25.  Increase  of  Susceptibility  of  Yeast  by  Exposure  to  S02_ 61 

26.  Weakening  of  Yeast  by  Exposure  to  S02 63 

27.  Micro-organisms  found  on  Grapes  from  Davis 66 

28.  Micro-organisms  found  on  Grapes  from  Contra  Costa  County 67 

29.  Micro-organisms  found  on  Grapes  from  Acampo 68 

30.  Number  of  micro-organisms  in  various  Musts 68 

31.  Relative  Stability  of  Liquid  and  Solid  Yeast  Cultures ,    71 

32.  Correspondence  of  Balling  per  cent  and  Number  of  Yeast  Cells 87 

33.  Fermentations  with  Starters  of  various  Balling  per  cent 78-79 

34.  Times  for  filling  and  using  Defecating  and  Yeast  Vats 84 

35.  Effect  of  S02  and  Pure  Yeast  on  Micro-organisms 87 

36.  Fermentation  of  Zinfandel  from  Acampo 93 

37.  Fermentation  of  Zinfandel  from  Acampo 94 

38.  Fermentation  of  Zinfandel  from  Acampo  with  sulfite  and  pure  yeast 94 

39.  Fermentation  of  Zinfandel  from  Acampo  with  sulfite  and  pure  yeast 95 

40.  Fermentation  of  Zinfandel  from  Acampo  with  sulfite  and  pure  yeast 95 

41.  Summary  of  Zinfandel  Fermentations 96 

42.  Fermentation  of  Petite  Sirah  from  Acampo 99 

43.  Fermentation  of  Petite  Sirah  from  Acampo  with  sulfite 99 

44.  Fermentation  of  Petite  Sirah  from  Acampo  with  sulfite  and  pure  yeast —  99 

45.  Summary  of  Petite  Sirah  fermentations 100 

46.  Fermentation  of  Zinfandel  from  Contra  Costa  County 101 

47.  Fermentation  of  Zinfandel  from  Contra  Costa  County  with  sulfite  and  pure 

yeast   101 

48.  Fermentation  of  Zinfandel  from  Contra  Costa  County  with  sulfite  and  pure 

yeast   102 

49.  Fermentation  of  Green  Hungarian  from  Acampo 104 

50.  Fermentation   of  Green   Hungarian   from  Acampo   with   sulfite   and   pure 

yeast   104 


Page. 

51.  Fermentation   of   Green   Hungarian   from   Acampo   with   sulfite   and   pure 

yeast   104 

52.  Wines  showing  unfermented   Sugar 107 

53.  Influence  of  S02  on  Volatile  Acidity — Red  wines 109 

54.  Influence  of  S02  on  Volatile  Acidity — White  wines 109 

55.  Influence  of  S02  on  Fixed  Acidity 111 

56.  Influence  of  S02  on  Alcohol 112 

57.  Influence  of  S02  on  Color  of  Red  Wines 113 

58.  Sulfurous  Acid  remaining  in  the  Wine 114 

59.  Composition  of  Experiment  wines 115 


LIST  OF  DIAGRAMS. 

1.  Disappearance  of  S02  from  various  liquids 37 

2.  Disappearance  of  free  S02  from  various  musks 38 

3.  Disappearance  of  free  S02  from  raisin  must  and  from  fresh  must 39 

4.  Relation  of  Balling  degree  to  total  and  free  S02 41 

5.  Changes  in  total,  free  and  combined  S02  in  must  and  wine 42 

G.  Comparison  of  fermentative  activity  of  "trained"  and  "untrained"  yeast 62 

7.  Efficiency  of  Starters  of  various  Balling  per  cent 80 

8.  Typical  fermentation  curve  at  constant  temperature 90 

9.  Variations  in  temperature  of  fermenting  room 92 

10.  Fermentation  curves  of  Zinfandel  I,  II,  III,  IV,  V 98 

11.  Fermentation  curves  of  Petite  Sirah  VI,  VII,  VIII 100 

12.  Fermentation  curves  of  Zinfandel  IX,  X,  XI 102 

13.  Fermentation  curves  of  Green  Hungarian  I,  IV,  V,  VII,  IX 105 


LIST  OF  FIGURES. 

1.  Sulfur  hook,  cup  and  cage 29 

2.  Method  of  sulfuring  must  with  a  pump 30 

3.  Sulfur  machine 31 

4.  Device  for  spraying  must  into  sulfured  cask 33 

5.  Elimination  of  molds  by  S02 57 

6.  Apparatus  for  rejuvenation  of  pure  yeast 82 

7.  Incubator  for  pure  yeast 82 

8.  Yeast  propagating  apparatus  for  winery 83 


Digitized  by  the  Internet  Archive 

in  2012  with  funding  from 

University  of  California,  Davis  Libraries 


http://www.archive.org/details/enologicalinvest230biol 


EN0L06ICAL  INVESTIGATIONS 


I.    INTRODUCTION. 

Special  investigations  in  viticulture  and  enology  have  been  pursued 
by  the  Yiticultural  Division  of  the  College  of  Agriculture  in  fulfillment 
of  the  requirements  of  an  act  passed  by  the  legislature  of  the  State 
of  California  in  1909.  Details  of  the  purposes  of  this  act  are  given 
in  the  introduction  to  Bulletin  213.  In  accordance  with  the  provisions 
of  this  act  the  following  publications  have  been  issued : 

1.  Grape  Growing  in  the    Imperial    Valley.     In    Bulletin    210, 

January,  1911. 

2.  The  Principles  of  Wine-making.     Bulletin  213,  May,  1911. 

3.  The  Extermination  of  Morning-Glory.     Circular  69,   August. 

1911. 

4.  Hot-room  Callusing.     Circular  76,  August,  1911. 

The  legislature  of  1911,  confirming  the  act  of  1909,  continued  the 
appropriation  to  carry  out  its  provisions.  Two  publications,  including 
the  present  bulletin,  have  been  issued  as  the  result. 

5.  Grape  Vinegar.     Bulletin  227,  January,  1912. 

6.  Enological  Investigations.     Bulletin  230,  June,  1912. 

The  principal  improvements  in  the  art  of  wine-making  contributed 
by  the  last  twenty-five  years  are  the  accurate  and  intelligent  use  of 
sulfurous  acid,  the  application  of  pure  yeast  to  the  fermentation  of 
grapes  and  the  control  of  the  temperature  in  every  stage  of  the  process. 

The  proper  use  of  sulfurous  acid  has  made  it  possible  to  avoid  com- 
pletely the  production  of  spoiled  wines.  No  wine-maker  who  under- 
stands this  use  and  applies  his  knowledge  has  any  vinegar-sour,  lactic, 
mousey,  slimy,  or  otherwise  diseased  wines  in  his  cellar.  High  volatile 
acid,  unfermented  sugar,  persistent  cloudiness,  defects  due  in  nearly  all 
cases  to  bacterial  growth  can  be  completely  eliminated.  It  enables 
him  to  have  every  gallon  of  his  wine  sound,  dry  and  clear  within  three 
months  or  less  from  the  crushing  of  the  grapes.  This  is,  therefore,  the 
first  and  most  important  improvement  in  his  art  that  the  wine-maker 
should  study. 

The  application  of  pure  yeast  is  perhaps  the  next.  It  is  the  com- 
plement of  the  use  of  sulfurous  acid.  It  may  not  in  every  case  enable 
him  to  improve  his  best  wine,  but  it  will  give  much  greater  certainty 
to  his  operations  and  undoubtedly  improve  the  average  quality  and 
increase  the  uniformity  of  his  product.     It  prevents  those  strange  varia- 


24  UNIVERSITY   OF    CALIFORNIA EXPERIMENT    STATION. 

tions  of  quality  between  casks  of  wine  made  from  the  same  grapes  by 
the  same  methods  which  have  so  long  puzzled  wine-makers. 

The  control  of  temperature  is  the  final  improvement  which  gives 
him  absolute  control  over  every  detail  of  the  wine-making  process.  In 
addition  to  the  two  other  means,  it  enables  him  to  reduce  his  opera- 
tions to  an  exact  method  and  to  eliminate  chance.  By  use  of  all 
three,  he  can  lay  out  his  work  with  a  precision  and  certainty  unknown 
to  the  old  forms  of  wine-making.  By  restricting  the  rise  of  tempera- 
ture during  the  violent  fermentation  and  maintaining  it  during  the 
after  fermentation  the  delicacy  and  the  finer  qualities  of  the  wine  can 
be  greatly  improved.  Refrigerating  the  wine  to  a  suitable  degree  after 
the  elimination  of  the  sugar  promotes  its  rapid  clarification  by  the 
precipitation  and  deposition  of  salts,  micro-organisms  and  the  other 
solid  matters  which  often  form  a  persistent  cloudiness  in  defectively 
handled  wines.  A  proper  rise  of  temperature  after  the  wine  has  been 
cleared  by  the  first  or  second  racking,  hastens  and  facilitates  the  matur- 
ing of  the  wine.  The  time  and  degree  of  this  rise  are  determined  by 
the  degree  of  aging  desired.  When  this  is  obtained,  a  lowering  of 
the  temperature  makes  it  possible  to  keep  the  wine  without  deprecia- 
tion until  it  can  be  brought  by  subsequent  finings,  rackings  and  other 
operations  into  the  condition  required  by  the  consumer. 

The  general  principles  and  practice  of  these  improvements  have  been 
discussed  in  Bulletin  167,  "The  Manufacture  of  Dry  Wines  in  Hot 
Countries";  Bulletin  213,  "The  Principles  of  Wine-making";  Circular 
22,  "Defecation  of  Must  for  White  Wine";  and  Circular  23,  "Pure 
Yeast  in  Wineries."  Experiment  work  on  their  applicability  to  Cali- 
fornian  conditions  has  been  reported  in  Bulletin  174,  "A  New  Wine- 
cooling  Machine,"  and  Bulletin  177,  "A  New  Method  of  Making  Dry 
Red  Wine." 

Cooling  devices  have  been  installed  in  a  large  number  of  Californian 
wineries  and  their  utility  is  very  generally  recognized  by  our  wine- 
makers.  Pure  yeast  has  been  used  with  success  for  several  years  in 
a  limited  number  of  wineries  in  the  State  and  would  become  more  gen- 
eral if  its  merits  and  ease  of  application  were  better  understood.  Sul- 
furous  acid  has  rapidly  come  into  very  general  use,  but  many  wine- 
makers  have  an  imperfect  understanding  of  its  effects  and  of  the  best 
methods  of  use. 

The  present  bulletin  is  an  attempt  to  popularize  the  last  two  improve- 
ments and  to  clear  up  some  uncertainties  regarding  their  use. 

Investigations  were  made  during  the  last  season  to  determine  to 
what  degree  the  injurious  micro-organisms  could  be  eliminated  and 
the  wine  yeast  encouraged  by  the  use  of  sulfurous  acid  and  pure  yeast 
and  to  discover  the  best  methods  and  doses  in  various  cases.     Some  of 


Bulletin  230]  ENOLOGICAL   INVESTIGATIONS.  25 

these  investigations  were  conducted  in  the  laboratory  and  some  at  the 
winery  of  Mr.  J.  E.  Colton.  who  very  obligingly  offered  us  the  neces- 
sary facilities.  Some  tests  were  made  on  a  scale  large  enough  to  dem- 
onstrate their  applicability  to  industrial  conditions  in  California,  and 
to  determine  how  much  improvement  could  be  expected  from  the  use 
of  sulfurous  acid  and  pure  yeast,  which  are  easily  and  cheaply  applied 
without  the  asistance  of  cooling  devices  which  require  more  trouble 
in  applying  and  more  expense  in  installing. 


II.    SULFUROUS  ACID  IN  WINE-MAKING. 

A.     General   Considerations. 

(a)  Historical. — The  use  of  sulfurous  acid  in  wine-making,  dating 
from  remote  antiquity  has  within  recent  times  undergone  remarkable 
developments. 

The  Romans  used  sulfur  to  mix  with  the  pitch,  wax,  incense  and 
spices  which  they  steeped  in  their  wine  or  burnt  in  the  vessels  in  which 
they  stored  it.  The  literature  of  more  modern  wine-making  contains 
numerous  favorable  references  to  the  utility  of  the  fumes  of  burning 
sulfur  for  the  treatment  of  casks  in  which  wine  was  to  be  kept.  Many 
authors  recommended  the  addition  to  the  sulfur  of  orris  root,  ginger  and 
similar  aromatic  substances.  Such  additions  were  doubtless  a  sur- 
vival of  the  ancient  practices  and  show  that  wine-makers  were  still 
ignorant  of  the  true  nature  of  the  action  of  sulfur  fumes. 

(b)  Modern  Developments. — Only  within  the  last  twenty-five  years 
have  we  begun  to  formulate  a  correct  theory  of  the  effects,  beneficial 
and  injurious,  of  "sulfuring, "  enabling  us  to  make  use  of  the  former 
while  avoiding  the  latter.  The  development  of  this  theory  we  owe  to 
the  general  progress  of  applied  science  and  especially  to  the  researches 
of  Nessler,  Weigert,  Strucchi,  Bouffard,  Martinand,  Laborde,  Wiley  and 
many  others  who  have  investigated  the  practice  from  the  chemical, 
industrial,  and  hygienic  sides. 

At  present,  sulfurous  acid  is  used  in  some  form  and  for  some  pur- 
poses in  every  winery  and  every  wine  cellar  where  good  wine  is  made 
and  where  anything  but  the  most  primitive  and  elementary  notions  of 
the  art  of  wine-making  exist. 

(c)  Opinions  of  Experts. — Babo  and  Mach,  in  the  last  edition 
(1910)  of  "  Kellerwirtschaf t, "  the  principal  work  on  wine-making  in 
the  German  language,  say:  "Sulfuring  is  indispensable."  "In  cellars 
where  the  practice  is  unknown,  it  is  hard  to  find  completely  sound  or 
merchantable  wines."  J.  Weinmann,  in  the  last  edition  of  "Manuel  du 
Travail  des  Vins  Mousseux,"  recommends  the  use  of  3  to  5  grams 


26  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

of  bisulfite  of  potash  per  hectoliter  of  must  in  the  manufacture  of 
champagne.  L.  Mathieu,  speaking  for  Burgundy,  says  (1911)  "Wine 
of  the  highest  quality  is  obtained  by  the  previous  sulfuring  of  the 
must."  J.  Laborde,  discussing  the  preparation  of  the  white  wines  of 
the  Gironde,  states,  (1910),  "The  use  of  sulfurous  acid  in  white  wine 
is  a  benefit — even  a  necessity."  E.  Dupont,  giving  the  results  of  his 
observations  on  the  use  of  sulfurous  acid  in  the  south  of  France,  says 
(1910),  "The  practice  should  become  a  part  of  the  general  method  of 
wine-making,"  and  further,  "We  are  obliged  to  recognize  that  since 
the  use  of  sulfites  in  the  fermenting  vats  has  become  general,  the  num- 
ber of  badly  made,  defective  or  diseased  wines,  formerly  so  frequent, 
has  diminished  considerably;  in  fact,  such  wines  have  almost  disap- 
peared from  commerce."  Finally,  at  the  International  Congress  of 
Madrid  (1911)  five  resolutions  were  adopted  having  reference  to  the 
improvement  of  wine  and  its  methods  of  manufacture.  Three  of 
these  resolutions  speak  in  favorable  terms  of  the  use  of  sulfurous  acid 
and  the  other  two  recommend  methods  which  are  based  on  its  use. 

Among  modern  authorities  there  are  but  few  dissenting  opinions, 
and  these  come  from  chemists  or  medical  men  who  have  little  or  no 
knowledge  of  wine,  and  whose  views  are  based  on  observations  of  the 
action  of  sulfurous  acid  in  cases  where  the  objects  and  effects  are 
totally  different  from  those  of  its  proper  use  in  wine-making. 

It  may  be  taken  as  demonstrated,  therefore,  that  to  obtain  the  best 
wine  from  the  point  of  view  both  of  the  producer  and  of  the  consumer, 
the  rational  use  of  sulfurous  acid  is  necessary.  No  substitute  has  yet 
been  found  of  which  the  efficiency  has  been  demonstrated.  There  are 
serious  objections  to  all  which  have  been  proposed  and  none  of  them 
accomplish  all  the  desirable  objects  attained  by  the  use  of  sulfurous 
acid. 

(d)  Legal  Limitations. — In  order  to  obtain  these  results,  however, 
it  is  necessary  that  the  wine-maker  should  understand  clearly  how  the 
sulfurous  acid  acts  and  the  limits  within  which  it  must  be  used.  A 
lack  of  this  understanding  on  the  part  of  the  wine-maker  and  a  confu- 
sion of  the  effects  of  the  free  acid  with  those  of  the  combined  acid,  as 
it  occurs  in  wine,  on  the  part  of  earlier  investigators  has  led  to  legal 
limitations  on  its  use.  These  limitations  were  at  first  so  narrow  as 
practically  to  prohibit  its  use,  but  with  increasing  knowledge  of  the 
facts,  they  have  been  gradually  widened  until  they  are  now  sufficient 
to  cover  all  the  amounts  which  the  intelligent  wine-maker  needs.  The 
present  tendency  of  pure  food  laws  seems  to  be  to  place  the  limitations 
at  about  .035  per  cent  or  350  milligrams  per  liter  of  total  sulfurous 
acid  of  which  not  more  than  70  milligrams  may  be  free.  There  is  no 
need  ever  to  exceed  these  limits,  and  the  wine-maker  who  does  so  will  in 


Bulletin  230]  ENOLOGICAL   INVESTIGATIONS.  27 

most  cases  injure  the  quality  and  merchantable  value  of  his  wine  irre- 
spective of  the  legal  limitations.  For  the  protection  of  the  consumer 
there  is  little  need  of  legal  limitations,  as  an  excess  which  approaches 
an  amount  harmful  to  the  consumer  will  so  depreciate  the  selling  value 
of  the  wine  that  the  abuse  would  tend  to  cure  itself.  The  establish- 
ment of  legal  limits,  however,  has  been  of  great  use  to  the  wine-maker 
by  making  it  necessary  for  him  to  study  carefully  the  methods  of  apply- 
ing sulfurous  acid,  and  of  graduating  the  amount  to  the  case  in  hand 
in  order  to  obtain  the  maximum  beneficial  effects. 

B.      Properties,  Preparation,  Use  and   Effects  of  Sulfurous  Acid. 

(a)  Properties. — Sulfurous  acid  (S02)  is  a  colorless  gas  2.2  times 
as  heavy  as  air,  easily  recognized  by  its  characteristic  odor.  Sulfur 
itself  has  no  odor  and  the  so-called  "sulfur  smell"  is  due  to  the  S02 
produced  when  sulfur  is  burned. 

The  gas  is  soluble  in  water  in  the  proportion  of  30  to  45  volumes  at 
ordinary  temperatures.  At  the  temperature  of  20°  C.  one  volume  of 
water  will  dissolve  36.4  volumes,  or  one  pound  of  water  will  dissolve 
.104  pounds  of  the  gas.  The  gas  liquefies  at  ordinary  atmospheric  pres- 
sure when  its  temperature  is  lowered  to  — 10°  C.  At  the  temperature 
of  20°  C.  it  exerts  a  pressure  of  3.25  atmospheres  or  40.6  pounds  per 
square  inch. 

Sulfurous  acid  gas  will  not  burn  like  hydrogen  or  illuminating  gas 
nor  support  combustion  like  air  or  oxygen. 

It  has  a  bleaching  action  on  organic  colors  which  is  sometimes  utilized 
in  decolorizing  fabrics,  nuts,  dried  fruits,  etc.  It  owes  this  property  to 
its  power  of  forming  colorless  compounds  by  combining  with  the  color- 
ing matters.  These  compounds  can  usually  be  broken  up  by  oxidation 
and  the  color  restored.  Pink  must  or  wine  can  be  made  white  by  treat- 
ment with  S02,  but  on  aeration  the  color  returns.  The  bleaching  action 
thus  differs  materially  in  character  from  that  of  chlorine  which  destroys 
the  color,  or  that  of  charcoal  which  removes  it. 

When  dry  it  is  inactive.  In  the  disinfection  of  rooms,  the  air  is 
first  saturated  with  moisture  by  means  of  steam  which  allows  the  S02 
to  exert  its  germicidal  properties. 

Compounds  are  readily  formed  by  S02  with  certain  organic  sub- 
stances called  aldehydes  of  which  formaldehyde  and  acetaldehyde  are 
typical.  The  latter  occurs  in  wine  together  with  others  similar  in  very 
small  quantities.  These  compounds  are  what  are  known  as  the  "com- 
bined" form  of  S02  as  it  occurs  in  wine.  This  property  has  been 
utilized  for  the  removal  of  S02  from  wine  by  the  addition  of  urotropine. 
This  substance  acts  by  breaking  up  slowly  with  the  evolution  of  for- 


28  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

maldehyde.  As  the  latter  is  harmful,  the  process  is  useless  and  illegal 
when  applied  to  wine. 

Similar  combinations  take  place  between  S02  and  the  sugar  of  grape 
must.  These  combinations  are  fairly  stable  and  they  seem  to  constitute 
the  main  part  of  the  combined  S02  in  grape  must. 

When  introduced  into  wine  or  grape  must,  some  of  the  S02  fails 
to  enter  into  any  of  these  combinations.  It  is  this  part  which  is  called 
the  "free"  S02.  It  is  this  form  which  has  the  active  antiseptic  and 
germicidal  properties  utilized  in  wine-making.  It  is  also  this  form 
which  in  excess  is  harmful  to  animals  and  men  who  consume  excess- 
ively sulfured  products,  and  to  plants  growing  in  the  vicinity  of  fac- 
tories which  give  off  "sulfur  fumes." 

In  solution  in  water,  it  readily  attacks  alkalies,  metallic  oxides  and 
most  metals,  forming  salts  termed  sulfites,  bisulfites,  and  metabisulfites, 
according  to  the  proportion  of  S02  which  they  contain.  Dissolved  in 
water,  therefore,  it  is  a  true  acid.  Its  acid  properties  are  weak,  that 
it  to  say,  it  is  readily  liberated  from  its  salts  by  other  acids  such  as 
sulfuric,  tartaric,  citric,  and  acetic.  This  explains  the  odor  of  burning 
sulfur  given  off  by  a  fermenting  vat  when  the  grapes  have  been  treated 
with  metabisulfite.    The  S02  is  set  free  by  the  tartaric  acid  of  the  must. 

(b)   Preparation  and  Use  in   Wineries:     L  sulfur  fumes. — When 
sulfur  is  burned  in  the  air,  oxygen  is  taken  up  and  sulfurous  acid  gas 
is  formed.    The  chemical  reaction  involved  is  expresed  thus : 
S      +      2  0       =S02. 
Sulfur +2  Oxygen = Sulfurous  acid. 

This  is  the  reaction  which  takes  place  when  a  cask  is  disinfected 
by  burning  a  piece  of  sulfur  tape.  Sulfur  wicks  or  matches  are  made 
by  coating  strips  of  asbestos  or  of  thin  linen  or  cotton  cloth  or  of  tough 
paper  with  sulfur.  The  fabric  is  cut  into  strips  about  1J  inches  wide 
and  10  or  12  inches  long.  These  strips  are  then  dipped  into  melted 
sulfur  kept  at  the  lowest  temperature  at  which  it  will  remain  liquid. 
By  dipping  two  or  three  times  and  allowing  the  sulfur  to  harden 
between  times,  a  thick  coating  of  sulfur  can  be  obtained.  As  the  burn- 
ing of  the  cloth  core  produces  ill-smelling  fumes  the  smaller  the  pro- 
portion of  cloth  to  sulfur  the  better. 

When  used  for  sulfuring  small  casks,  these  wicks  are  usually  sus- 
pended by  means  of  a  sulfur  bung  and  burnt  in  the  cask.  A  sulfur 
bung  consists  of  a  wooden  bung  sufficiently  long  and  tapering  to  fit  all 
sizes  of  bung  holes,  and  furnished  with  a  piece  of  iron  wire  10  inches 
to  15  inches  long  inserted  in  the  bottom  of  the  bung  at  one  end  and 
turned  up  to  form  a  sharp  hook  at  the  other. 


Bulletin 


:m] 


EXOLOGICAL   INVESTIGATIONS. 


29 


There  are  several  serious  objections  to  using  sulfur  in  this  way.  Part 
of  the  sulfur  melts  and  falls  to  the  bottom.  If  it  burns  there,  it  injures 
the  staves  of  the  cask,  if  it  fails  to  burn  it  may  communicate  a  bad  taste 
to  the  wine.  Part  volatilizes  and  is  deposited  as  fine  flowers  of  sulfur 
on  the  walls  of  the  cask  where  it  may  later  be  changed  into  hydrogen 
sulfide  by  the  action  of  yeast.  Hydrogen  sulfide  is  the  cause  of  the 
1 'rotten  egg"  smell  of  some  wines  which  is  the  same  as  that  familiar 
at  sulfur  springs.  Another  portion  of  the  sulfur  forms  evil-smelling 
compounds  with  the  cloth  core  of  the  wick  and  still  another  portion  is 
oxidized  to  sulfuric  acid,  S03. 

To  prevent  the  falling  of  the  molten  sulfur  to  the  bottom  of  the 
cask  the  sulfur  wick  may  be  burned  in  a  "sulfur  cage,"  consisting  of 
a  narrow  metal  or  earthenware  cylinder  with  perforations  on  the  sides 


a  b  c 

Fig.  1. — Devices  for  burning  sulfur  in  casks. 
a,  sulfur  hook ;  b,  sulfur  cup  ;  c,  sulfur  cage. 

to  admit  air.  This  cage  has  a  solid  bottom  to  catch  the  melting  sulfur 
and  is  suspended  in  the  cask  by  means  of  a  sulfur  bung.  Sulfur  cups 
of  similar  design  are  made  in  which  the  sulfur  can  be  burnt  directly 
and  the  defects  due  to  burning  cloth  obviated.  The  latter  are  not  very 
convenient  to  use,  however,  except  where  very  small  quantities  of  sulfur 
are  needed.  In  sulfuring  large  casks  furnished  with  a  manhole  at  the 
bottom,  the  sulfur  is  usually  burned  in  an  iron  or  earthenware  pan  on 
the  bottom  of  the  cask.  This  pan  should  be  supported  by  a  brick  or 
other  means  of  protecting  the  staves  from  the  heat  of  the  burning 
sulfur. 

Various  devices  for  sulfuring  are  in  use  in  which  the  sulfur  is  burned 
outside  of  the  cask.     One  of  these  consists  of  a  barrel  from  which  one 


30  UNIVERSITY  OF   CALIFORNIA EXPERIMENT   STATION. 

head  has  been  removed.  It  is  inverted  over  a  pan  in  which  the  sulfur 
is  burned.  Air  is  admitted  through  holes  near  the  ground  and  the 
sulfur  fumes  introduced  into  the  must  or  wine  by  means  of  an  air 
pump  connected  with  the  top  of  the  barrel  and  the  cask  to  be  sulfured. 
The  sulfur  fumes  quickly  corrode  a  metal  pump.     See  Fig.  2. 

A  better  type  of  sulfurmg  machine  and  one  in  more  common  use 
is  illustrated  in  Fig.  3.  It  consists  of  a  stove  in  which  the  sulfur  is 
burned  connected  with  a  tall  cylindrical  vessel  in  which  the  sulfur 
fumes  are  brought  into  intimate  contact  with  the  must.  The  heated 
gas  and  air  entering  the  bottom  of  the  cylinder  pass  over  the  surface  of 
the  must,  which  flows  over  a  series  of  inclined  plates  and  the  whole 
o  _- ^ 


Fig.  2. — Method  of  sulfuring  must  with  a  pump. 

M,  small  cask  with  one  head  removed,  serving  as  a  chamber  in  which  to  burn  the 
sulfur ;  f,  f,  openings  for  entrance  of  air ;  a,  r,  hose  through  which  the  sulfur 
fumes  are  forced  into  the  cask  of  wine  by  means  of  the  pump,  P. 

of  the  S02  produced  is  absorbed.  By  interposing  a  water  trap  between 
the  stove  and  the  cylinder,  and  forcing  the  fumes  through  this  trap 
by  means  of  an  air  blast,  the  volatilized  sulfur  and  sulfuric  acid  may 
be  removed. 

This  device  removes  most  of  the  objections  cited  above,  but  the 
extreme  uncertainty  of  measuring  and  the  difficulty  of  regulating  the 
amount  of  S02  introduced  into  the  must  still  exist. 

A  device  for  partially  removing  these  objections  consists  of  a  turbine 
turned  by  the  must  which  flows  over  it.  This  turbine  operates  a  fan 
which  supplies  the  air  to  the  sulfur  stove.  As  the  rapidity  of  the 
turbine  varies  with  the  rate  of  flow  of  the  must,  and  the  S02  produced 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


31 


varies  with  the  amount  of  air  passing  through  the  stove,  a  means  of 
regulating  the  amount  of  S02  in  accordance  with  the  volume  of  must 
is  afforded. 

With  even  the  most  perfect  of  these  devices,  one  other  serious  defect 
of  the  method  of  obtaining  S02  directly  from  burning  sulfur  remains. 
This  is  the  extreme  uncertainty  of  the  amount  of  S02  produced  by 
burning  a  given  weight  of  sulfur. 

According  to  the  formula  of  the  reaction,  one  part  by  weight  of  sulfur 


Fig.  3. — Sulfur  machine. 


on  burning  should  yield  two  parts  by  weight  of  S02,  or  one  ounce 
should  yield  .782  cubic  feet  of  the  gas  at  60°  F. 

In  practice  the  yield  is  much  less  than  this.  Pacottet  states  that  the 
quantity  of  S02  actually  formed  when  sulfur  is  burned  in  a  small  cask 
varies  from  15  per  cent  to  60  per  cent  of  that  corresponding  to  the 
amount  of  sulfur  consumed.  This  loss  is  due  to  the  sulfur  which  is 
volatilized  or  melted  without  burning  and  to  that  which  forms  sul- 
furic acid.    It  varies  with  the  amount  of  sulfur  burnt  in  a  given  space 


32 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


and  with  the  mechanical  condition  of  the  sulfur.  When  there  is  a  full 
supply  of  air  and  when  the  sulfur  is  well  exposed  to  it,  the  combustion 
is  more  perfect.  As  the  air  becomes  exhausted  of  its  oxygen,  com- 
bustion becomes  less  perfect.  When  all  the  sulfur  possible  is  burned 
in  a  closed  space,  a  greater  proportion  is  lost  than  if  a  smaller  quan- 
tity is  burned. 

When  only  a  very  small  quantity  of  S02  is  needed,  as  in  racking 
wine,  and  careful  methods  of  burning  are  used,  the  weight  of  sulfur 
may  be  taken  as  a  fair  measure  of  the  S02  produced  which  may  be  as 
high  as  85  per  cent*  of  the  theoretical  amount.  When  larger  quantities 
are  needed,  the  weight  of  sulfur  used  becomes  a  very  uncertain  measure 
of  the  S02  obtained.  When  the  larger  quantities  are  needed,  as  in  the 
defecation  of  must,  it  is  interesting  to  know  what  is  the  maximum 
amount  of  S02  that  can  be  produced  in  a  cask  by  burning  sulfur.  By 
using  an  excess  of  sulfur  that  lost  by  volatilization  and  melting  does 
not  come  into  the  account. 

A  cask  or  other  closed  space  contains  a  certain  amount  of  oxygen 
which,  if  it  combined  with  sulfur  would  produce  exactly  twice  its 
weight  of  S02.  This  we  will  call  the  theoretically  possible  yield  which 
is  about  .6  grams  to  1,000  c.c.  of  air  at  0°  C.  In  practice,  the  possible 
yield  is  much  less  than  this.  The  difference  is  caused  by  losses  of 
oxygen,  some  of  which  combines  to  form  sulfuric  acid,  some  escapes 
from  the  barrel  as  the  air  expands  with  the  rise  of  temperature  and 
some  fails  to  combine  when  the  proportion  in  the  air  becomes  too  small 
to  support  the  combustion  of  the  sulfur. 

These  facts  are  shown  by  the  results  of  tests  given  in  the  following 
table : 

TABLE  No.   1. 
S02  produced  by  burning  sulfur  in  a  closed  space. 


Volume  of  vessel. 

Sulfur 
used. 

Sulfur 
melted. 

Sulfur 
sublimed. 

S02  formed. 

Sulfur 
utilized. 

Oxygen 
utilized. 

1.    3700  c.c.    __    

.180 

.520 

.600 

1.300 

0.0 

0.0 
.08 
.56 

1.68 

.0494 
.1790 
.1880 
.3050 
.2150 

.2613 

.6830 
.8241 
.8710 
.7900 

72.6% 

65.7 
68.7 
33.5 
14.2 

11.6% 

30.4 

2.    3700  c.c 

3.    3700  c.c. 

36.6 

4.    3700  c.c.  _. 

38.7 

5     3700  c.c.  _ 

2.290 

351 

Test  No.  1  represents  the  burning  of  a  little  more  than  6  ounces  of 
sulfur  in  a  1,000  gallon  cask.  Nearly  three  fourths  of  the  sulfur  is 
oxidized  to  S02  and  about  one  tenth  of  the  oxygen  of  the  air  is  utilized. 
As  we  increase  the  amount  of  sulfur  used  we  gradually  approach  a 
maximum  production  of  S02  which  corresponds  to  about  1.5  pounds  to 
2  pounds  of  sulfur  in  a  1,000  gallon  closed  cask  and  utilizes  a  little 


*  Laborde. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


33 


more  than  one  third  of  the  oxygen  present.  If  we  use  more  than  this 
maximum  the  excess  of  sulfur  simply  melts  or  sublimes  and  no  more 
SO.,  is  produced  unless  we  renew  the  air  in  the  cask. 

Another  element  of  uncertainty  in  this  method  of  sulfuring  is  the 
failure  of  the  must  to  take  up  all  the  S02  produced  in  the  cask.  Some 
of  it  always  escapes  with  the  air  forced  out  of  the  cask  by  the  incoming 
must.  This  loss  is  increased  with  the  rapidity  of  filling,  it  is  greater 
when  the  must  is  introduced  at  the  bottom  of  the  cask  and  when  it 
enters  in  a  solid  stream.  It  is  greater  usually  in  small  casks  than  in 
large.  It  can  be  diminished  by  filling  the  cask  slowly  and  especially 
by  introducing  the  must  in  a  spray  by  means  of  a  rose-nozzle  or  similar 
device. 


Fig.  4. — Device  for  spraying  must  into  sulfured  cask. 

R,  hose  for  entrance  of  wine  ;  T,  cover  of  manhole  to  which  is  attached  by  cords  a 
small  piece  of  wood,  P,  on  which  the  entering  wine  strikes  and  is  broken  up  into 
a  spray. 

In  small  casks  it  can  be  almost  eliminated  by  first  introducing  a 
small  quantity  of  must,  5  per  cent  to  10  per  cent  of  the  total,  and  then 
rolling  the  cask  until  all  the  S02  has  been  absorbed  and  filling  with  the 
rest  of  the  must. 

An  idea  of  the  amount  of  loss  by  failure  of  all  the  oxygen  to  com- 
bine to  form  SOo  and  by  escape  of  a  portion  of  that  formed  is  given 
by  the  following  experiments.  In  wooden  barrels  of  various  sizes  and 
in  glass  flasks  holding  2,600  c.c.  all  the  sulfur  possible  was  burned. 
Each  was  then  filled  with  water,  must  or  wine,  run  in  slowly.  As  soon 
as  full,  the  amount  of  S02  in  the  liquid  in  each  vessel  was  determined 
with  the  results  shown  in  Table  2. 


2—230 


34 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


TABLE    No.    2. 
Absorption  of  S02  with  maximum  sulfuring. 


Container. 


Per  cent  S02 

Theoretical 

Liquid. 

absorbed. 

yield. 

Water 

.00796 

.0546 

Water 

.00948 

.0546 

Water 

.00920 

.0546 

Must 

.00535 

.0546 

Must 

.00504 

.0546 

Must 

.00663 

.0546 

Wine 

.0154 

.0546 

Water 

.0133 

.0546 

Water 

.0126 

.0546 

Water 

.0142 

.0546 

Water 

.0168 

.0546 

Water 

.0132 

.0546 

Water 

.0118 

.0546 

Ratio  to 

theoretical 

yield. 


2600  c.c.  flask 

2600  c.c.  flask 

2600  c.c.  flask 

2600  c.c.  flask 

2600  c.c.  flask 

2600  c.c.  flask 

2600  c.c.  flask 

5  gallon  barrel 
5  gallon  barrel 
28  gallon  barrel 
28  gallon  barrel 
28  gallon  barrel 
50  gallon  barrel 


14.38% 

17.34 

16.85 

9.79 

9.23 
12.14 
28.51 
24.17 
23.07 
26.01 
30.76 
24.17 
21.43 


From  Table  No.  2  it  is  seen  that  wine  absorbs  the  gas  more  readily 
than  water  and  water  in  turn  is  a  better  absorber  of  S02  than  must. 
The  averages  for  each  in  a  2,600  c.c.  bottle  are :  water  88.8  milligrams, 
S02  per  liter,  must  56.7  milligrams  per  liter  and  wine  154  milligrams 
per  liter.  The  per  cent  of  sulfurous  acid  absorbed  in  the  barrels  was 
greater  on  the  average  than  in  the  glass  bottle,  owing  no  doubt  to  the 
relatively  greater  surface  exposed  in  the  wooden  containers,  due  to  the 
inequalities  of  the  surface  and  pores  of  the  wood.  The  film  of  water 
in  the  pores,  etc.,  absorbs  the  S02  rapidly.  The  figures  indicate  that 
by  burning  a  maximum  amount  of  sulfur  in  small  barrels  and  running 
in  the  liquid  by  means  of  a  plain  hose  the  must  or  wine  will  be  sul- 
fured  at  the  rate  of  50  to  150  milligrams  per  liter. 

We  may  reckon  roughly  that  where  all  ordinary  precautions  are 
taken  to  make  the  must  absorb  all  the  S02  produced  by  burning  as 
much  sulfur  as  possible  in  a  cask,  we  obtain  as  a  maximum  one  third 
of  the  amount  corresponding  to  the  oxygen  of  the  air.  This  represents 
a  sulfuring  of  approximately  200  milligrams  of  S02  per  liter  or  .02  per 
cent,  corresponding  to  an  addition  of  40  grams  of  potassium  meta- 
bisulfite  to  a  hectoliter  or  about  3  pounds  to  1,000  gallons  of  must.  This 
is  about  the  minimum  amount  necessary  in  the  defecation  of  must  and 
requires  the  burning  of  about  2  pounds  of  sulfur  in  a  1,000  gallon  cask. 

2.  Liquid  Sulfurous  Acid. — Gaseous  S02  can  be  liquefied  by  pres- 
sure or  by  cooling  below  — 10°  C,  at  which  temperature  it  remains 
liquid  at  ordinary  atmospheric  pressure.  The  liquid  is  1.4  times  as 
heavy  as  water.  It  vaporizes  rapidly  at  ordinary  temperatures  unless 
confined  under  presure. 

Liquid  S02  is  manufactured  commercially  in  Germany  from  crude 
sulfur  or  iron  pyrites.  The  gas  obtained  by  burning  the  former  or 
roasting  the  latter  is  purified  by  passing  through  water,  which  retains 


Bulletin  230]  ENOLOGICAL   INVESTIGATIONS.  35 

such  impurities  as  sulfuric  acid,  arsenic,  sublimed  sulfur  and  zinc  sul- 
fate. The  gas  is  then  cooled  a  few  degrees  below  0°  C.  to  get  rid  of 
the  water  vapor  and  finally  liquefied  by  pressure.  The  pure,  dry  liquid 
is  then  placed  in  iron  cylinders  for  shipment. 

When  used  in  wine-making,  some  measuring  device  is  necessary.  In 
that  constructed  by  Pacottet,  the  liquid  is  forced  by  the  pressure  in  the 
cylinder  into  a  small  glass  measuring  tube  from  which  it  is  allowed  to 
vaporize,  through  a  small  copper  tube,  directly  into  the  must  or  wine. 

The  advantages  of  this  method  of  sulfuring  are  the  great  accuracy 
with  which  the  doses  can  be  measured,  the  non-corrosive  properties  of 
the  pure,  dry  liquid  and  the  absence  of  such  impurities  as  sulfuric 
acid,  free  sulfur  and  hydrogen  sulfide  which  accompany  the  S02 
obtained  directly  from  burning  sulfur. 

3.  Solutions  of  Sulfurous  Acid.  S02  is  soluble  in  water,  which  dis- 
solves about  69  volumes  at  0°  C.  The  gas  is  lost  rapidly  with  a  rise 
of  temperature  and  at  20°  C.  water  will  hold  only  36  times  its  volume 
and  at  40°  C.  less  than  19.  This  variability  of  the  water  solution 
makes  it  entirely  unsuited  for  use  in  wine-making  and  its  corrosive 
action  on  metals  and  its  bulkiness  make  it  inconvenient  to  handle.  It 
is  produced  cheaply  as  a  by-product  of  smelters  and  in  some  cases 
might  be  used  as  a  disinfectant. 

S02  is  more  soluble  in  alcohol,  and  the  solutions,  therefore,  less  bulky. 
They  are,  however,  also  too  variable  and  unstable  for  use  in  wine  and 
too  expensive  for  use  in  disinfection. 

4.  Solid  Forms;  Salts.  Sulfurous  acid  unites  with  bases  such  as 
potash,  soda  and  lime  to  form  various  series  of  salts  of  which  the  prin- 
cipals are  the  sulfites,  bisulfttes,  and  metabisulfites.  These  salts  all 
break  up  with  the  evolution  of  S02  when  they  are  brought  in  contact 
with  a  stronger  acid.  The  tartaric  acid  of  grape  must,  for  example, 
will  combine  with  the  base  and  liberate  the  S02,  which  is  then  free  to 
exert  its  action  on  the  must. 

The  base  in  combination  with  the  tartaric  acid  remains  in  the  must 
or  wine.  For  this  reason  the  soda  and  lime  salts  cannot  be  used,  as 
they  would  introduce  something  foreign  to  the  grapes. 

The  potash  salts  on  the  other  hand,  introduce  only  bitartrate  of 
potash  or  cream  of  tartar,  a  normal  ingredient  of  the  wine.  The 
amount  introduced,  moreover,  is  very  small,  much  less  than  the  varia- 
tions between  different  samples  of  grapes.  Any  of  the  pure  potash 
salts  may  be  used,  but  they  differ  considerably  in  strength,  cost  and 
convenience. 

In  potassium  sulfite,  K2S03,  the  potash  is  combined  with  the  mini- 
mum amount  of  S02,  of  which  it  contains  approximately  41  per  cent. 
The  bisulfite,  KHS03,  contains  when  pure,  53  per  cent  of  S02,  but  it 


36 


UNIVERSITY  OF   CALIFORNIA — EXPERIMENT   STATION. 


loses  strength  rapidly  by  oxidation.  The  metabisulfite,  K2S205,  is  the 
strongest  of  all,  containing  when  perfectly  pure  and  fresh,  a  little 
over  57  per  cent.  It  is  also  more  stable  than  the  bisulfites.  As  found 
in  commerce,  it  contains  only  from  50  per  cent  to  54  per  cent  and  in 
practice  it  is  usual  to  consider  that  the  salt  will  yield  half  its  weight 
in  S02,  which  is  exact  enough  for  ordinary  usages.  It  will  keep  fairly 
constant  in  composition  for  months  or  even  years  if  kept  in  tightly 
stoppered  glass  vessels  and  in  a  dry  place.  For  use  in  wine-making,  it 
should  be  guaranteed  free  from  all  injurious  impurities. 

It  is  applied  directly  in  the  solid  form  to  the  must  or  made  up 
shortly  before  using  into  a  10  per  cent  solution  in  water.  Water  solu- 
tions must  be  made  and  kept  in  glass  or  earthenware  vessels,  as  when 
concentrated  they  act  very  rapidly  on  metals. 

(c)  Transformations  of  Sulfurous  Acid.  The  S02  which  is  intro- 
duced into  the  must  or  wine  does  not  all  remain  free  or  active.  Some 
of  it  unites  with  the  sugar  and  other  components  of  the  liquid  and 
becomes  more  or  less  inactive  in  the  combined  form.  Finally,  both 
the  free  and  the  combined  forms  disappear  more  or  less  completely 
and  with  more  or  less  rapidity.  The  "total  S02"  includes  both  the 
free  and  the  combined. 

1.  disappearance  from  various  solutions.  To  ascertain  the  extent 
and  rate  of  disappearance  from  various  solutions  the  following  tests 
were  made : 

TABLE   No.   3. 

Disappearance  of  S02  from  various  solutions. 


Solution. 

Time. 

S02  per  liter 

Solution. 

Time. 

S02  per  liter 

Water   

0 

674  m.g. 
630 

Water,     plus      .5% 
tartaric  acid. 

0 

674  m.g. 
542 

50  hrs. 

50  hrs. 

92 

541 

92 

395 

i 

169 

428 

169 

277 

r 

288 

302 

362 

68 

362 

231 

462 

40 

390 

186 

462 

138 

Water   

0 
26 

252 

176 

Water,      plus      .5% 
tartaric  acid. 

0 
26 

271 

207 

74 

25 

74 

143 

91 

0 

91 

134 

Fresh    must,    Ball- 

0 

200 

Raisin  must,  Ball- 

0 

200 

ing  20%. 

24 

185 

ing  24%. 

42 

197 

39 

165 

72 

192 

113 

125 

91 

166 

135 

113 

116 

134 

543 

103 

140 
193 

102 
96 

Raisin  must,  Ball- 

0 

600 

Raisin  must,  Ball- 

0 

1500 

ing  24%. 

18 
42 
67 

364 
320 
262 

ing  24%. 

42 

1075 

Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


37 


Some  of  the  results  of  Table  No.  3  are  shown  graphically  in  Diagram 
No.  1.  The  line  representing  the  disappearance  of  the  SO.,  from 
water  show  that  this  disappearance  is  very  rapid  and  almost  directly 
proportionate  to  the  time.  The  curve  of  the  line  of  the  solution  con- 
taining tartaric  acid  is  at  first  steep,  gradually  becoming  flatter.  This 
indicates  an  increase  in  the  rapidity  of  loss  of  S02  at  first  and  a  decrease 
later.  The  tartaric  acid  seems  at  first  to  facilitate  the  escape  of  SO, 
and  later  to  oppose  it, 

The  disappearance  is  much  slower  in  the  musts,  showing  a  retarding 
influence  due  to  the  various  substances  dissolved  in  it.  The  retarding 
influence  varies  in  the  different  musts.     The  raisin  must,  containing 


ILjxsa&BcamNccL 


iNwfjTm±s%mm 


&tu  ~sr?o 


Diagram  1    (see  table  3). 
Disappearance  of  S02  from  various  solutions. 


more  sugar  shows,  at  first,  slightly  more  retarding  effect  than  the  fresh 
must.  At  the  end,  however,  so  far  as  the  tests  were  carried,  the  raisin 
must  lost  slightly  more  of  its  S02. 

2.  disappearance  from  grape  must.  In  spite  of  the  superior  retard- 
ing effect  of  the  raisin  must,  it  commenced  to  ferment  sooner  than  the 
fresh  and  with  a  larger  total  amount  of  S02.  This  indicates  a  differ- 
ence in  the  fermentation  restraining  powTer  of  the  S02  in  the  two 
musts.  An  explanation  of  this  is  given  in  the  following  tests  showing 
the  changes  in  the  amounts  of  free  S02  in  various  musts. 

These  tests,  shown  graphically  in  diagram  2,  show  a  much  slower 
disappearance  of  the  active  free  S02  from  the  fresh  grape  must  than 
from  the  sweeter  raisin  must.  The  results  reduced  to  percentages  of 
free  S02  remaining  at  various  periods  are  compared  in  Table  5. 


38 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


TABLE  No.    4. 
Disappearance  of  free  S02  from  various  musts. 


Free  S02  in 

Experiment. 

Time  in  hours. 

milligrams 
per  liter. 

No.  215— Fresh  grape  must,  Balling  20% 

Added 

200 

0 

175 

24 

97 

39 

95 

113 

70 

135 

61 

168 

55 

543 

2.5 

No.  225— Raisin  must,  Balling  24%. 


No.  222— Raisin  must,  Balling  24%. 


No.  222— Raisin  must,  Balling  24%. 


No.  222— Raisin  must,  Balling  24% 


Added 

0 

18 

42 

67 

Added 

0 

18 

42 

67 


Added 

200 

0 

33 

1 

30 

6 

26 

25 

18 

56 

12 

80 

4 

Added 

400 

0 

100 

24 

35 

48 

35 

78 

32 

102 

14 

149 

7.8 

600 

116 

59 

46 

23 

1500 

269 

214 

128 

58 


ZO      ^O      GO 


do     /oo    tso    /4o  /eo 

Diagram  2  (see  Table  4). 


/SO   gOO  /5%0&fO 


Disappearance  of  free  SOs  from  various  musts. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


39 


TABLE   No.    5. 
Free  S02  remaining  in  fresh  and  raisin  must. 


Milli- 
grams 
per  liter 
added. 

Per  cent  remaining. 

On 

addition. 

36 

hours. 

67 
hours. 

113              168 
hours.         hours. 

1.  Fresh   must,   20%   Balling 

2.  Raisin  must,  24%  Balling 

3.  Raisin  must,  24%  Balling 

4.  Raisin  must,  24%  Balling 

5.  Raisin  must,  24%  Balling 

200 
200 
400 
600 
1500 

87.5 
16.5 
25.0 
19.3 
18.0 

47.5 
7.5 
8.8 
8.0 

10.0 

42.0 
4.0 
8.3 
3.9 
3.9 

35.0 

24.5 

The  amount  of  free  S02  in  the  raisin  must  seems  to  be  a  function  of 
the  amount  added  originally.  At  the  end  of  three  days  there  was  about 
the  same  per  cent  of  the  original  amount  present  in  the  must  which 
received  only  200  milligrams  per  liter  as  in  that  which  received  1,500 
milligrams.  The  fresh  must  at  the  same  time  showed  ten  times  as  much. 
That  the  free  S02  is  the  controlling  factor  in  the  delay  of  fermentation 
is  shown  by  the  fact  that  the  raisin  must  to  which  1,500  milligrams  was 
added,  started  to  ferment  sooner  than  the  fresh  must  which  received 
only  200  milligrams.  The  contrast  between  the  fresh  must  and  the 
raisin  musts  is  shown  clearly  in  diagram  3. 

JOO 
90 

80 

70 

eo 
so 

50 

zo 

10 


<$ 

^3 

T    I 

trf: 

\H  Mt 

fxr 

u\ 

W    i 

tW.*) 

IN 

a 

It  \ 

O 
ft- 

| 

IT 

HOU 

=?5 

o     30  40    eo 

Diagram   3    (see  Table   5).— 


dO      IOO    IZO    140    /eO    /80    ZOO  ZROZ40 

Disappearence    of  free   S02  from   raisin   must  and  from 
fresh  must. 


This  great  difference  might  be  due  to  the  power  of  some  constituent 
of  the  fresh  must  to  set  the  S02  free  or  of  some  constituent  of  the  raisin 
must  to  hold  it  in  combination.  Tests  showed  that  the  S02  was  held 
equally  well  in  the  raisin  must  when  the  acidity  was  raised  from  .61 
per  cent  to  1.01  per  cent,  and  little  difference  in  this  respect  could  be 
noticed  between  raisin  musts  of  19.5  per  cent  Balling  and  29  per  cent 
Balling.     The  neutralizing  power  of  the  raisin  must  was  found  to  be 


40 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


the  greater  the  higher  it  was  heated  before  the  addition,  and  is  appar- 
ently due  to  some  undetermined  substance  formed  from  the  raisin  must 
by  the  heating. 

Differences  of  neutralizing  powers,  similar  but  less  marked,  have 
been  noticed  in  practice  between  different  musts,  and  they  indicate 
one  of  the  reasons  for  the  need  of  using  larger  amounts  of  S02  in 
heavy  musts  made  from  grapes  growing  in  hot  regions  where  many 
dried  grapes  or  raisins  are  apt  to  occur. 

3.  winery  tests.  Observations  were  made  in  a  winery  to  see  how 
nearly  the  changes  in  form  and  amount  of  S02  found  in  laboratory 
tests  corresponded  with  the  actual  changes  in  practice. 

Three  vats  of  red  grapes  containing  approximately  10  tons  each 
were  tested  periodically.  No.  169  contained  Zinfandel  grapes  from 
Acampo  showing  22.5  per  cent  Balling,  No.  176,  the  same  grapes 
showing  22.3  per  cent  Balling  and  No.  179  Alicante  Bouschet  grapes 
from  the  same  locality  showing  22.5  per  cent  Balling. 


TABLE   No.    6. 
Disappearance  of  total  and  free  S02  from  red  wine  vats. 


Experiment. 

Time  in 
hours. 

Balling 
per  cent. 

Total  S02. 

Free  S02. 

No.  169— Zinfandel 

0.5 
49 
64 

22.5 
23.5 
23.5 

219 

127 

93 

90 

74 

23.6 

188 

90 

97 

2.5 

158 

50 

136 
181 

o 

0 

_ 

13 

126 

11 

No.  176— Zinfandel 

0.5 
120 

22.3 
0 

75 
66 

42 

19 

No.  179 — Alicante  Bouschet 

1 

7 

22.5 
22.0 

186 
169 

65 

54 

22 

19.5 

159 

36 

27 

15.0 

150 

30 

34 

10.0 

131 

30 

46 

3.5 

119 

30 

58 

0 

98 

30 

From  this  table  we  see  (Nos.  169,  179)  that  with  an  average  dose 
of  S02  for  red  wine  fermentation  (200  milligrams  per  liter  or  12  ounces 
of  metabisulfite  per  ton  of  grapes)  nearly  one  half  of  the  total  dis- 
appears during  fermentation.  With  smaller  additions  (No.  176)  a 
larger  proportion  remains. 

Reference  to  the  curves  of  diagram  4  shows  that  the  total  S02  dis^ 
appeared  rapidly  at  first  and  later  more  slowly  and  at  a  fairly  uniform 
rate.     The  free  S02  represents  at  first  about  35  per  cent  of  the  total, 


Bulletin  230] 


ENOUXilCAL    INVESTIGATIONS. 


41 


or  65  milligrams  per  liter,  falling-  in  about  seven  hoars  to  about  54 
milligrams  per  liter  at  which  point  fermentation  becomes  evident. 


im 

I6C 

Q-  £4 

.^*""""- 

ZQ7V 

[£_22< 

140 

—  a. 

^ZVAa 

120 

-J 

It    Id 

■r*-T/yi 

100 

fa 

flO 

CQ 

40 

^2S 

?<** 

3 

?,0 

/-/c 

£//?£ 

I 

>         I 

>       /< 

>      n 

J       x5 

o    2 

t>    a 

?     3 

3     40 

Diagram  4    (see  Table  6). 
Relation  of  Balling0  to  total  and  free  S02. 

4.  disappearance  from  wine.  The  changes  in  form  and  amount 
are  much  less  rapid  in  the  wine  than  in  the  must.  Table  No.  7  gives 
the  results  of  determinations  of  the  free  and  total  S02  made  at  intervals 
during  one  to  four  months  after  the  completion  of  fermentation.  The 
decrease  is  comparatively  small  in  all  cases.  In  some  cases  there  is  an 
increase.  This  is  due  to  racking  into  sulfured  casks  by  which  the 
supply  was  renewed. 

TABLE   No.    7. 
Disappearance  of  S02  from  wine  after  fermentation. 


Experiment. 

Time  days 

after 

fermentation. 

Total  S02 

milligrams 

per  1. 

Free  S02 

milligrams 

per  1. 

No.  169— Zinfandel  from  Acampo  . 

1 

4 

40 

66 

112 

0 

74 

120 

0 

60 
106 

2 

28 
74 

0 
46 

178 

170 

115 

96 

76 

66 
39 
40 

98 

96 

108 

155 

150 
151 

312 
340 

35 

No.  170— Zinfandel  from  Acampo 

33 

25 
26 

27 

19 

23 

No.  179— Alicante  Bouschet__ 

30 

No.  181— Green  Hungarian,  1500  gallon  tank 

No.  243— Palomino,  185  gallon  pnncheon__ 

33 

18 

14 
10 

13 

10 

15 

42 


UNIVERSITY  OP   CALIFORNIA — EXPERIMENT  STATION. 


5.  final  forms  of  so2.  The  change  in  the  form  and  amount  of  S02 
in  the  fermented  wine,  as  shown  in  table  7,  contrasts  noticeably  with 
those  which  take  place  in  the  must  before  and  during  fermentation  as 
shown  in  tables  4,  5  and  6. 

When  free  S02  is  added  to  must  in  the  quantities  usual  in  wineries 
either  in  the  form  of  gas,  liquid  or  sulfite  it  decreases  very  rapidly 
at  first.  The  total  S02  also  decreases,  but  with  less  rapidity.  The 
combined  S02,  on  the  contrary,  increases  rapidly  at  first,  the  rate  of 
increase  gradually  slackens,  then  ceases  and  finally  this  form  also 
decreases. 

The  courses  taken  by  the  two  forms  of  S02  are  shown  more  or  less 
schematically  by  diagram  5,  which  represents  an  average  red  wine 
fermentation  in  an  open  vat.  The  200  milligrams  per  liter  of  free  S02 
added  falls  rapidly  to  between  40  and  50  milligrams  in  about  two 
and  one  half  days,  when  evident  fermentation  starts.  The  point  in  time 
and  the  amount  of  free  S02  at  which  fermentation  starts  will  vary 
with  different  conditions  of  temperature  and  must  composition.  Dur- 
ing fermentation,  lasting  in  the  case  supposed  about  3  days,  the  free 
SO 2  continues  to  decrease  with  somewhat  diminishing  rapidity  until  it 
reaches  about  25  milligrams  per  liter  which  amount  remains  constant 
for  some  time. 

In  the  mean  while,  the  combined  S02  has  been  increasing  at  a  slightly 
slower  rate  than  the  decrease  of  the  free.  The  difference  between  the 
decrease  of  the  free  and  the  increase  of  the  combined  is  shown  by  the 
curve  indicating  the  net  decrease  of  the  total  S02. 

The  final  loss  of  S02,  shown  by  the  decrease  in  total,  takes  place 


zz  z* 


Diagram  5. — Changes  in  total,  free  and  combined  S02  in  must  and  wine.  A.  Ante- 
fermentation  period,  about  three  days.  B.  Fermentation  period,  about  three  days. 
C.  Post-fermentation  period. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


43 


in  two  ways.  Part  is  volatilized  and  escapes  into  the  atmosphere,  part 
is  oxidized  to  sulfuric  acid.  At  first  the  volatilization  is  rapid  and 
is  the  principal  cause  of  the  loss  shown  by  the  steepness  of  the 
curve.  The  escape  of  gaseous  S02  from  a  recently  sulfited  vat  is 
readily  recognized  by  its  strong  odor.  During  fermentation  the 
decrease  becomes  slower,  owing  probably  to  a  slackening  or  cessation 
of  the  vaporization.  After  fermentation,  the  decrease  is  further  dimin- 
ished owing  to  the  cessation  of  vaporization  and  a  diminution  in  the 
rate  of  oxidation.  In  the  wine  after  fermentation,  the  decrease  of 
total  and  of  combined  S02  is  identical.  This  probably  means  that  as 
fast  as  the  free  S02  is  oxidized,  it  is  renewed  by  the  setting  free  of 
an  equivalent  amount  of  the  combined  forms. 

The  curves  of  diagram  5  simply  represent  general  or  typical  forms 
which  may  vary  in  detail  in  different  cases.  For  example,  the  change 
from  the  free  to  the  combined  form  will  be  more  rapid  in  musts  from 
overripe  grapes.  The  rapidity  of  loss  of  total  S02  may  be  much 
greater  than  indicated  if  the  must  is  fermented  in  small  vats  with 
abundant  aeration,  and  it  may  be  much  slower  if  fermented  in  closed 
casks  of  large  size.  The  nature,  and  causes  of  some  of  these  variations 
are  discussed  later. 

The  final  form  of  all  the  S02  which  does  not  escape  into  the  atmos- 
phere is  sulfuric  acid.  The  acid  thus  produced  acts  on  the  cream  of 
tartar  giving  potassium  sulfate  and  free  tartaric  acid  in  small  amounts. 
A  measure  of  the  increase  in  sulfates  due  to  this  cause  is  given  by 
the  following  tables  based  on  laboratory  and  winery  tests. 


TABLE  No.   8. 
Increase  of  sulfates  due  to  use  of  S02.      (Laboratory  tests.) 


Experiment. 

S02  used 

milligrams 

per  1. 

K2S04. 

Increase  of 
K2S04. 

K2S04 

corresponding 

to  total 

S02  added. 

No.  138— Sultanina 

0 

200 

.0259 
.0421 

.0162 

.0544 

200 

.0396 

.0137 

.0544 

300 

.0597 

.0338 

.0816 

300 

.0550 

.0291 

.0816 

Sultanina* __ ___ 

0 
10 

.0218 
.0230 

.0012 

.0027 

50 

.0314 

.0096 

.0136 

75 

.0358 

.0140 

.0204 

100 

.0388 

.0170 

.0272 

150 

.0572 

.0354 

.0408 

200 

.0678 

.0460 

.0544 

250 

.0766 

.0548 

.0680 

'Bettoli.   R.  W.     Thesis,  1911. 


44 


UNIVERSITY   OF    CALIFORNIA EXPERIMENT   STATION, 


The  increase  of  sulfates  is  shown  to  be  approximately  proportional 
to  the  amounts  of  S02  used,  but  is  in  all  cases  less  than  the  amount 
corresponding  to  its  total  oxidation.  Even  when  large  amounts  are 
used,  (300  milligrams  per  liter),  the  increase  is  not  sufficient  to 
approach  the  legal  limit  of  2  grams  per  liter. 

Analyses  of  wines  fermented  with  S02  in  the  winery  gave  similar 
results. 

TABLE   No.    9. 
Increase  of  sulfates  clue  to  the  use  of  S02.      (Winery  tests.) 


Experiment. 

S02  used, 
milligrams 
per  liter. 

Sulfates  in 

unsulfited 

wine. 

Sulfates  in 
sulfited 
wine. 

No.  243— Palomino _    _ 

600 

187 
185 
185 
100 

.0790 
.0527 
.0692 
.0446 
.0225 

.0887 

No.  169 — Acampo  Zinfandel  __ _  _ 

.0548 

No.  176— Zinfandel    

.0682 

No.  181 — Green  Hungarian 

.0564 

No.  173— Petite  Sirah «      _      

.0749 

These  tests  show  that  the  variations  in  the  sulfates  of  different 
grapes  and  in  different  samples  of  the  same  grape  are  much  greater 
than  the  increases  due  to  the  use  of  sulfurous  acid. 

In  the  case  of  the  Green  Hungarian  musts,  the  original  amount  of 
total  S02  was  185  milligrams  per  liter.  At  the  time  of  analysis  it  was 
approximately  150  milligrams  per  liter,  indicating  a  loss  of  35  milli- 
grams per  liter.  The  increase  in  sulfates  over  the  untreated  wine  was 
.0118  grams  K2S04  equivalent  to  .00305  grams  of  sulfurous  acid  or 
to  30  milligrams  per  liter.  This  indicates  that  most  of  the  S02  in  a 
white  wine  fermentation  disappears  by  being  oxidized  to  the  sulfate  of 
potash.  On  the  other  hand,  with  the  red  wine,  e.  g.,  the  Acampo  Zin- 
fandel, only  a  very  small  amount  of  the  sulfurous  acid  is  oxidized  to 
the  sulfate,  indicating  that  most  of  it  disappears  as  sulfur  dioxide  gas 
or  is  precipitated  in  the  lees  as  gypsum;  that  is,  sulfate  of  lime.  The 
latter  is  probably  the  case,  for  when  the  wines  were  examined  after 
racking,  that  is,  six  weeks  after  the  above  analyses  the  sulfates  had 
decreased  very  appreciably  in  all  the  wines.  A  few  figures  w7ill  show 
this  plainly. 

TABLE  No.   10. 
Loss  of  sulfates  in  wine  after  fermentation. 


Experiment. 

k2so3. 

December  19, 
1911. 

February  3, 
1911. 

Loss, 
per  cent. 

No.  171— Zinfandel  (no  sulfite)  _ 

.0527 

.0548 
.0692 
.0749 
.0571 
.0571 
.0608 

.0343 
.0590 
.0505 
.0590 
.0559 
.0530 
.0560 

34.0 

No.  169— Zinfandel   (sulfited) 

—  7.7 

No.  176— Zinfandel  (sulfited) 

27.1 

No.  173— Petite  Sirah  (sulfited)--. 

21.5 

No.  179 — Alicante  Bouschet  (sulfited) 

2.2 

No.  182 — Green  Hungarian  (no  sulfite)  __    _  __ 

7.0 

No.  181 — Green  Hungarian  (sulfited)  _-  _ 

8.0 

Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


45 


(d)  Influence  of  Composition  and  Treatment  of  Must  on  Fermenta- 
tion Inhibiting  Power  of  802.  Laboratory  tests  have  shown  that  fer- 
mentation does  not  start  in  must  to  which  sulfurous  acid  has  been 
added  until  the  quantity  of  free  S02  has  fallen  to  an  amount  which  is 
somewhere  between  30  and  50  milligrams  per  liter. 

This  reduction  in  the  amount  of  free  S02  is  due  to  its  combination, 
vaporization  and  oxidation.  It  is  more  or  less  complete  and  proceeds 
with  more  or  less  rapidity  according  to  the  character  of  the  must  and 
to  the  treatment  to  which  the  must  is  subjected.  Tests  were  made  to 
determine  the  influence  of  various  factors  on  this  reduction.  The 
factors  investigated  were  the  acid  and  sugar  contents,  the  presence  of 
pomace  and  other  solid  matters,  the  degree  of  ripeness  of  the  grapes 
and  the  heating  of  the  must  to  various  temperatures. 

The  tests  were  conducted  in  a  series  of  8  ounce  bottles.  In  each 
bottle  was  placed  100  c.c.  of  must,  sulfited  to  the  required  degree  with 
a  2  per  cent  water  solution  of  potassium  metabisulfite.  The  bottles, 
after  inoculation  with  1  c.c.  of  a  vigorous  culture  of  Burgundy  wine 
yeast,  were  kept  at  a  temperature  of  30°  C.  and  the  start  of  fermenta- 
tion determined  by  the  commencement  of  the  evolution  of  bubbles 
of  gas. 

1.  acidity.  To  determine  the  effect  of  variations  in  acidity,  a  must 
was  used  containing  1.18  per  cent  of  total  acidity  as  tartaric.  In 
series  a  the  acidity  was  reduced  to  .217  per  cent  by  the  addition  of 
water,  in  series  b  to  .6  per  cent  by  the  same  means.  In  series  c  the 
acidity  was  reduced  to  .63  per  cent  by  means  of  caustic  potash.  Series 
d  (check)  received  must  without  any  addition  and  in  series  e  the 
acidity  was  increased  to  1.8  per  cent  by  the  addition  of  free  tartaric 
acid.  In  all,  the  sugar  content  was  made  equal  to  that  of  the  check 
by  means  of  glucose. 

TABLE   No.    11. 
Influence  of  acidity  of  must  on  effect  of  S02. 


Experiment. 

Acidity. 

Number  of  hours  before  the  start  of  fermentation. 
S02  added  in  milligrams  per  liter. 

100. 

150. 

300. 

500. 

a             _ 

.217 

.600 

.630 

1.180 

1.800 

24  hours 
24  hours 
48  hours 
69  hours 
48  hours 

69  hours 

69  hours 

69  hours 

120  hours 

192  hours 

192  hours 

240  hours 

69  hours 

* 

336  hours 

576  hours 
* 

b 

c 

96  hours 
* 

d 

e 

576  hours 

*No  fermentation. 


A  diminution  of  the  effect  of  S02  with  a  decrease  in  acidity  is 
plainly  indicated  by  a  comparison  of  the  check  d  with  a  and  b,  in  which 
the  acidity  had  been  reduced  by  dilution  with  water.    This  diminution 


46 


UNIVERSITY  OF   CALIFORNIA — EXPERIMENT  STATION. 


is  much  increased  in  series  c  where  the  acidity  was  reduced  by  neutral- 
ization with  caustic  potash.  In  series  a  and  b  the  ratio  between  the 
free  tartaric  and  the  bitartrates  to  which  the  acidity  is  due  is  the  same 
as  in  the  check  series  d.  In  series  c  the  free  tartaric  had  been  changed 
in  whole  or  part  to  bitartrate.  That  indicates  that  free  tartaric  acid 
intensifies  the  effect  of  the  S02  and  explains  in  part  the  need  of  larger 
amounts  in  overripe  grapes  in  which  little  free  tartaric  acid  exists. 
Some  other  factor,  perhaps  experimental  error — appears  to  have  influ- 
enced series  e,  where  the  addition  of  free  tartaric  does  not  show  an 
intensification  of  the  effect  of  S02  except  in  the  150  milligram  column. 

2.  sugar.  The  influence  of  the  sugar  contents  of  the  must  on  the 
effect  of  S02  was  tested  in  another  must. 

To  a  series  of  four  flasks  containing  must  of  18.1  per  cent  Balling 
was  added  100,  150,  300  and  500  milligrams  per  liter  of  S02.  In  a 
parallel  series  the  must  was  raised  to  22  per  cent  Balling  and  in  another 
to  26  per  cent  Balling  by  the  addition  of  grape  sugar.  No  differences 
were  noted  in  the  time  of  starting  fermentation.  The  greater  resist- 
ance of  must  made  from  very  ripe  grapes,  therefore,  does  not  seem  to 
depend  on  the  larger  sugar  contents. 

3.  limpidity.  The  influence  of  the  presence  in  the  must  of  undis- 
solved solid  matters  was  next  investigated.  Series  a  represents  a  red 
wine  fermentation  in  which  the  must  ferments  in  contact  with  the 
solid  parts  of  the  grape.  Series  c  represents  a  clear  must  such  as  might 
be  obtained  from  clean,  sound  grapes.  Series  b  represents  a  must 
from  inferior  grapes  or  from  a  continuous  press. 

table  No.  12. 
Influence  of  solid  matters  of  the  grape  on  effect  of  S02. 


Experiment. 

Number  of  hours  before  start  of  fermentation. 
S02  added,  milligrams  per  liter. 

100. 

150. 

300.                       500. 

a. 

Must  and  skins 

26 
26 
26 

26 
26 
26 

26 

240 

* 

144 

b. 

Cloudy  must 

* 

c. 

Filtered  must  __ 

* 

♦No  fermentation. 

This  shows  that  the  presence  of  the  skins  very  much  diminishes  the 
effect  of  S02  and  indicates  that  more  should  be  used  in  the  fermenta- 
tion of  red  wine  than  of  white.  In  a  cloudy  must  the  effect  is  also 
slightly  less  than  in  a  clear  one  and  shows  one  reason  for  the  use  of 
larger  quantities  with  must  from  moldy  or  damaged  grapes. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


47 


4.  heating.  The  heating  of  the  must  or  grapes  before  fermentation 
is  practised  to  a  limited  extent,  and  the  following  table  shows  the 
influence  of  various  degrees  of  heating  on  the  effect  of  S02.  The  same 
must  was  used  in  the  four  series,  which  were  heated  to  the  degrees 
indicated  and  then  kept  at  30°  C.  The  must  was  heated  and  cooled 
before  the  addition  of  the  S02. 


TABLE  No.   13. 

Influence  of  the  heating  of  must  on  the  effect  of  S02. 


Temperature 

of    Heating. 

Number  of  hours  before  start  of  fermentation. 
S02   added  in   milligrams  per  liter. 

100. 

150. 

300.                   500. 

30°  C. 

24 
24 
46 
26 

69 
46 
46 
26 

120 
* 

320 
26 

* 

80°  C,  15  min. 

* 

100°  C,  60  min 

* 

120°  C,  60  min 

500 

*No  fermentation. 

There  seems  to  be  no  evidence  here  that  heating  to  100°  C.  has  any 
influence  on  decreasing  the  effect  of  the  S02.  It  seems,  on  the  con- 
trary, to  have  tended  to  delay  the  fermentation.  This  may  have  been 
due  to  the  expulsion  of  the  dissolved  oxygen  which  was  perhaps  insuf- 
ficiently replenished  by  subsequent  aeration.  Heating  under  pressure 
to  120°  C.  for  an  hour,  however,  had  a  noticeable  influence  on  decreas- 
ing the  effect  of  S02,  added  subsequently,  resembling  in  a  smaller 
degree  the  effect  of  raisins  shown  in  Tables  6  and  14. 


48 


UNIVERSITY  OP   CALIFORNIA— EXPERIMENT   STATION. 


5.  ripeness.  The  next  tests  show  the  influence  of  various  degrees 
of  ripeness.  Series  a  and  b  contained  musts  made  from  somewhat 
underripe  grapes,  series  c  must  from  ripe  grapes  and  series  d  a  must 
made  from  raisins. 

TABLE   No.    14. 
Influence  of  degree  of  ripeness  of  grapes  on  effect  of  SO?. 


Must. 

S02  added, 
milligrams 
per  liter. 

Hours  before  start  of 
fermentation. 

More  than. 

Less  than. 

(a)    Unripe  Rose  of  Peru 

0 

50 
100 
150 
200 
300 

0 

50 
100 
150 
200 
300 

0 
50 
100 
150 
200 
250 

0 

200 

400 

600 

800 

1000 

1500 

2000 

0 

25 

48 

70 

188 

188 

0 

24 
26 
26 
46 
120 

25  hrs. 

(b)    Unripe  Tokay  __  

46 

70 

188 

280 

280 

24 

(c)    Ripe  Sultanina _ 

26 

46 

46 

120 

280 

20 

23 

44 

71 

92 

139 

(d)     Raisins  (dried  Zinfandel) _      

24 

24 

24 

24 

24 
24 
24 
43 

43 
43 
43 
75 

This  table  shows  marked  variations  in  the  influence  of  different 
grapes  in  the  effectiveness  of  the  S02.  In  the  Rose  of  Peru  must  50 
milligrams  delayed  the  start  of  the  fermentation  as  much  as  150  milli- 
grams in  the  Tokay  must.  The  difference  between  the  Tokay  and  the 
Sultanina  must  is  not  great.  The  influence  of  the  raisin  must  in 
counteracting  the  effect  of  the  S02  is  remarkable.  An  addition  of  600 
milligrams  had  no  apparent  effect  on  the  start  of  fermentation  and 
1,500  milligrams  had  less  effect  than  150  milligrams  in  any  of  the 
other  musts.  Even  2,000  milligrams  delayed  the  fermentation  only 
about  50  hours,  which  is  much  less  than  the  effect  of  200  milligrams  in 
the  other  musts. 

These  tests  show  that  to  obtain  a  certain  effect  in  wine-making  by 
the  use  of  S02,  the  amount  necessary  to  use  varies  very  much  with 
various  conditions.     Very  ripe  grapes  require  from  two  to  three  times 


Bulletin  230] 


ENOLOGICAL  INVESTIGATIONS. 


49 


as  much  as  moderately  ripe  or  underripe  grapes  while  grapes  con- 
taining raisins  may  require  even  larger  amounts.  About  twice  as  much 
is  necessary  in  the  manufacture  of  red  wine  where  the  must  ferments 
in  the  presence  of  the  skins  as  in  white  wine  where  the  must  ferments 
alone.  Musts  from  moldy  or  dirty  grapes  or  very  cloudy  musts  from  a 
continuous  press  require  more  than  clear  musts  carefully  extracted 
from  clean,  sound  grapes.  The  heating  of  the  must  seems  to  have 
little  effect,  unless  excessive,  in  which  case  it  may  necessitate  the  use 
of  slightly  larger  amounts  of  S02. 

(e)     Effects  of  S02   on  the  Micro-organisms  of  Must   and   Wine. 

1.    COMPARISON  OF  THE  EFFECTS   OF  FREE  AND   OF   COMBINED   S02.       It  IS  a 

recognized  fact  that  the  S02  which  exists  free  in  the  must  is  more 
active  in  its  effects  on  micro-organisms  than  the  combined.  To  obtain 
data  on  the  relative  efficiency  of  the  two  forms,  pure  cultures  of  a  wine 
yeast  were  exposed  to  various  amounts  of  free  and  to  various  amounts 
of  combined  S02  for  a  period  of  twenty-four  hours.  The  yeast  was 
then  transferred  to  flasks  of  must  containing  no  S02,  and  note  taken 
of  the  cases  in  which  fermentation  started.  Where  fermentation  oc- 
curred the  yeast  had  evidently  not  been  killed,  where  it  did  occur  the 
yeast  had  been  killed  or  rendered  incapable  of  developing  under  the  con- 
ditions of  the  experiment. 

The  free  S02  was  obtained  by  adding  various  amounts  of  a  solution 
of  metabisulfite  to  water  in  which  the  yeast  was  placed.  The  combined 
form  was  obtained  by  adding  various  amounts  of  the  solution  of  meta- 
bisulfite to  raisin  must  and  allowing  this  must  to  stand  until  most  of 
the  S02  had  combined  before  adding  the  yeast. 


TABLE  No.    15. 

Comparison  of  effects  of  free  with  those  of  combined  S02. 


Exposure  to  free  S02,  milligrams  per  liter. 

Exposure  to  combined  S02,  milligrams  per  liter. 

On  addition 

of  yeast, 

total. 

After 

24  hours, 

total. 

On  addition  of  yeast. 
Combined.        Free. 

After  24  hours. 

Combined.        Free. 

d. 
e_ 
f- 


27 

23 

50 

40 

100 

92 

245 

220 

481 

452 

926 

858 

h 

i- 
j- 

k 

! 


250 

4 

250 

652 

11 

594 

809 

14 

716 

1019 

17 

979 

1286 

22 

1181 

1804 

31 

1804 

4 

10 
12 
17 
20 
31 


After  an  exposure  for  twenty-four  hours  to  the  quantities  of  S02 
indicated  in  the  table,  the  yeast  of  each  flask  was  removed  to  flasks  of 
must  where  the  conditions  were  made  as  favorable  as  possible  to  fer- 
mentation. 
3—230 


50 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


The  yeast  from  flask  d  produced  fermentation  but  those  from  flasks 
e  and  f  did  not.  This  indicates  that  an  exposure  for  twenty-four  hours 
to  between  250  and  500  milligrams  of  free  S02  per  liter  will  kill  yeast 
or  render  it  incapable  of  development.  On  the  other  hand,  the  yeasts 
in  all  the  series  from  g  to  I  containing  combined  S02  fermented  without 
difficulty,  indicating  that  1,800  milligrams  of  combined  S02,  even  when 
accompanied  with  a  small  quantity  of  free,  has  no  effect  on  the  vitality 
of  the  yeast. 

A  closer  approximation  to  the  relative  efficiency  of  the  two  forms 
was  made  by  further  tests  of  the  same  character  shown  in  Table  16. 

TABLE  No.    16. 
Comparison  of  effects  of  free  with  those  of  combined  S02. 


Exposure  to  free  S02,  milligrams  per  liter. 


Exposure  to  combined  S02,  milligrams  per  liter. 


On  addition 

of  yeast, 

total. 

After 

24  hours, 

total. 

Amount 

added, 

total  S02. 

24  hours  after 
addition  of  yeast. 

Combined. 

Free. 

a 

253 
301 
354 

424 
477 
555 

178 
230 

250 
307 
477 
550 

g _ 

2000 

3000 
4000 
5000 
6000 
7000 

1426 
2365 
3080 
3894 
4146 
5113 

118 

b  __ 

h 

277 

c                     _ 

i 

j 

fc 

330 

d 

542 

e 

896 

f 

I 

826 

The  yeast  from  flask  b,  which  contained  over  230  milligrams  of 
free  S02,  fermented  while  that  of  flask  c,  which  contained  less  than 
354  milligrams,  did  not.  The  amount  of  free  S02  which  will  perma- 
nently paralyze  yeast  in  twenty-four  hours  under  the  conditions  of  the 
experiment,  therefore,  is  less  than  354  milligrams  and  more  than  230 
milligrams. 

The  yeast  from  flask  h,  containing  over  2,365  milligrams  of  combined 
S02,  fermented,  while  that  of  flask  i,  containing  less  than  4,000  milli- 
grams, did  not.  The  flasks  with  combined  S02,  however,  contained  also 
smaller  amounts  of  free  which  must  be  taken  into  account  in  estimat- 
ing the  relative  efficiency  of  the  two  forms  from  these  figures.  Flask 
h,  which  fermented,  contained  277  milligrams  of  free  S02.  If  we  take 
354  milligrams  as  the  amount  of  free  necessary  to  kill  the  yeast  as 
indicated  by  flask  c,  we  may  reasonably  conclude  that  free  S02  cor- 
responding to  the  difference  between  this  figure  and  that  of  277,  the 
amount  of  free  S02  in  flask  h,  is  more  effective  than  the  2,365  milli- 
grams of  combined  in  the  latter  flask.  The  relative  efficiency  of  the 
two  forms  in  killing  yeast  seems  to  be,  therefore,  somewhere  near 
77/2365.  This  indicates  that  the  free  S02  is  more  than  thirty  times 
as  effective  in  this  respect  as  the  combined.    This  figure,  however,  must 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


51 


not  be  considered  as  anything  more  than  an  approximation.  It  is 
quite  possible  that  the  true  ratio  in  this  respect  is  very  much  larger. 
All  that  the  tests  prove  is  that  under  some  conditions  about  300  milli- 
grams of  free  S02  will  kill  yeast,  and  that  combined  S02  in  much 
larger  amounts  than  are  ever  used  in  practice  has  no  permanent  injuri- 
ous effect  on  it. 

The  object  of  the  foregoing  tests  was  to  determine  the  permanent 
effect  of  S02  on  yeast  which  had  been  exposed  to  its  action.  As  was 
shown,  an  exposure  to  about  300  milligrams  per  liter  will  kill  yeast  in 
twenty-four  hours.  This  may  be  taken  as  a  measure  of  its  disinfectant 
power.  Its  antiseptic  power  measured  by  the  amount  in  a  nutritive 
solution  which  will  prevent  fermentation  is  much  smaller.  The  ratio 
in  antiseptic  effectiveness  between  the  free  and  combined  is  similar 
to  that  in  disinfectant  effectiveness  as  shown  by  the  following  tests. 

Wine  yeast  was  introduced  into  raisin  must  containing  2,000  milli- 
grams per  liter  of  total  S02.  Determinations  of  the  free  S02  were 
made  at  intervals  and  the  time  of  the  start  of  fermentation  noted. 

TABLE  No.   17. 
Amount  of  free  S02  which  will  permit  fermentation  to  start. 


Date. 

Total  S02  added             Free  S02  found 
milligrams  per  liter.       milligrams  per  liter. 

Remarks. 

January   1 
January  10 

a 

2000 

b 

2000 

a 

246 
83 
66 
32 

b 
246 

83 

Added  yeast. 
No  fermentation. 

January  17 

No  fermentation. 

January  25 

39 

A  fermenting;  B  not  fermenting. 

In  this  test  the  yeast  did  not  become  active  until  the  amount  of 
free  S02  had  fallen  to  between  30  and  40  milligrams  per  liter.  This  is 
probably  not  a  maximum  for  wine  yeast,  as  the  long  exposure  to  large 
quantities  of  free  S02,  which  occurred  at  the  beginning  of  this  test, 
probably  weakened  the  yeast  and  delayed  its  start. 

To  compare  the  antiseptic  effect  of  combined  S02  a  parallel  test 
was  made.  A  series  of  flasks  was  filled  with  a  raisin  must  which 
had  been  heated  to  a  high  temperature  under  pressure  to  cause  rapid 
combination  of  the  S02  added  later.  From  200  to  2,000  milligrams 
were  added  to  the  different  flasks  which  were  then  inoculated  with  1 
per  cent  of  a  vigorous  culture  of  wine  yeast.  All  the  flasks  commenced 
to  ferment  within  three  days.  This  shows  that  2,000  milligrams  per 
liter  of  S02  will  not  prevent  fermentation,  if  most  of  it  is  in  the  com- 
bined form  and  indicates  an  antiseptic  ratio  of  over  60  to  1  in  favor 
of  the  free. 


52 


UNIVERSITY   OF   CALIFORNIA EXPERIMENT   STATION. 


2.  EFFECT  OF  S02  ON  THE  MULTIPLICATION  OF  THE  PRINCIPAL  MICRO- 
ORGANISMS occurring  on  grape.  One  of  the  chief  uses  of  sulfurous 
acid  in  wine-making  is  to  prevent  the  development  and  activity  of 
injurious  organisms  entering  the  fermenting  vats  with  the  grapes  or 
from  other  sources.  Experiments  were  made  to  determine  the  relative 
susceptibility  of  the  principal  of  these  as  compared  with  wine  yeast. 

Five  series,  each  of  4  small  flasks,  were  filled  with  normal  grape 
must  and  to  each  flask  of  a  series  a  different  amount  of  S02  was  added. 
Each  series  was  then  inoculated  with  a  pure  culture  of  a  different 
organism  and  the  number  of  active  cells  determined  by  means  of  plate 
cultures.  Thirty-six  hours  later  the  number  of  active  cells  in  each 
flask  was  determined  again  in  the  same  way. 

TABLE   No.    18. 
Effect  of  S02  on  multiplication  of  micro-organisms.* 


S02  milligrams 
per  liter. 


Wine  yeast. 


Apiculatus. 


Wild  yeast.     ! 

Pastorianus        Penicillium. 
form. 


Aspergillus. 


Vinegar 
bacteria. 


Number  of  cells  at  start. 


20,000 


150,000 


620,000 


120,000 


450,000 


310,000 


Number  of  living  cells  after  thirty-six  hours. 


0 

50 

100 

200 

400 


640,000 

2,000,000 

310,000 

36,000 


200,000 

75,000 

56,000 

0 


580,000 

6,000 

190 

0 


40,000 
0 
0 
0 


120,000 

20,000 

30,000 

0 


610,000 

14,000 

300 

2 

0 


*A11  the  organisms  except  the  wine-yeast  were  isolated  from  California  grapes. 

This  table  shows  very  clearly  the  superior  resistance  of  wine  yeast. 
Fifty  milligrams  per  liter  diminished  the  number  of  molds  and  bacteria 
and  allowed  only  a  very  small  increase  of  apiculatus.  The  numbers 
of  the  last  were  decreased  by  100  milligrams.  The  wine  yeast  increased 
rapidly  with  50  and  100  milligrams  and  with  slightly  less  rapidity  with 
200  milligrams.  Even  with  400  milligrams  its  increases  were  greater 
than  that  of  apiculatus  with  only  50  milligrams. 

These  tests  indicate  that  with  the  must  used  and  under  the  condi- 
tions of  the  experiment,  an  addition  of  100  milligrams  per  liter  (equal 
to  26  ounces  of  metabisulfite  to  a  thousand  gallons)  the  development 
and  activity  of  all  the  common  injurious  micro-organisms  would  be 
prevented  and  a  practically  pure  yeast  fermentation  insured. 

3.    EFFECT    OF    S02    ON    THE    MIXTURE    OF    MICRO-ORGANISMS    OCCURRING 

naturally  on  grapes.  When  grapes  are  brought  to  the  winery,  and 
are  crushed,  large  numbers  of  molds  and  wild  yeasts  adhering  to 
their  surfaces  get  into  the  must.  Some  true  wine  yeast  is  usually 
present  on  ripe  grapes  but  in  smaller  numbers.     Experiments  were 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


53 


made  to  test  the  effect  of  small  additions  of  S02  on  such  mixtures  of 
micro-organisms.  A  must  from  somewhat  moldy  and  damaged  Palo- 
mino grapes  was  used,  containing  consequently  a  large  number  of 
active  cells.  The  must  was  divided  among  a  number  of  flasks  to  each 
of  which  a  different  small  quantity  of  metabisulfite  was  added. 

Before  adding  the  S02,  a  count  was  made  of  the  number  of  active 
cells  present.  Three  hours  after  the  addition,  another  count  was 
made.  Before  taking  the  samples  on  which  the  counts  were  made,  the 
flask  was  thoroughly  agitated  so  that  the  count  represents  the  total 
number  of  active  cells  present,  none  of  them  having  been  removed  by 
settling. 

TABLE  No.   19. 
Modification  of  relative  numbers  of  micro-organisms  in  natural  must. 


S02  added, 
milligrams  per  liter. 

Time  exposed. 

Number  of  active  cells  per  cubic  centimeter. 

Molds. 

Apiculatus.             Yeast. 

0 

0 

3  hrs. 

3 

o 

<-> 

3 
3 

97,625 
13,310 
19,360 
14,650 
7,600 
4,800 

7,341,400               58.575 

30  

189,930 

21,780 

14,650 

0 

0 

182,995 

75 

33,880 

120  

43,950 

180 

58,400 

240 

73,600 

This  shows  that  the  relative  susceptibility  of  the  molds,  apiculatus 
and  wine  yeast  are  the  same  in  mixtures  as  in  pure  cultures.  The 
reduction  in  the  proportions  of  active  undesirable  cells  is  very  marked 
and  very  rapid  even  with  small  amounts.  In  the  untreated  must  the 
apiculate  yeast  constituted  98  per  cent  of  the  total  number  of  cells 
and  the  wine  yeast  less  than  1  per  cent.  In  three  hours  after  the  addi- 
tion of  75  milligrams  per  liter  (equal  to  5  ounces  of  metabisulfite  per 
ton)  this  proportion  was  changed  to  28  per  cent  for  the  apiculate  and 
45  per  cent  for  the  wine  yeast.  With  180  milligrams  per  liter  (equal 
to  12  ounces  of  metabisulfite  per  ton)  the  apiculatus  was  eliminated 
and  the  wine  yeast  constituted  88  per  cent  of  the  total.  The  small 
proportion  of  active  mold  spores  left  is  negligible,  as  they  usually 
have  no  effect  on  the  fermenting  wine. 

A  winery  test  of  a  similar  nature  gave  similar  results.     Zinfandel 

grapes,  which  had  been  transported  by  rail,  were  crushed  into  a  2,200 

gallon  fermenting  vat  and  sulfited  at  the  rate  of  8  ounces  of  potassium 

metabisulfite  to  the  ton.    A  counting  of  the  active  cells  was  made  before 

sulfiting    and    another    an    hour    after    the    addition  of  S02.     In  the 

untreated  must  wTas  found: 

Molds 1,600,000  per  cubic  centimeter  =  35.7  per  cent. 

Apiculatus  2,830,000  per  cubic  centimeter  =  63.2  per  cent. 

Wild  yeasts 30,000  per  cubic  centimeter  =      .7  per  cent. 

Wine  yeast 20,000  per  cubic  centimeter  =      .4  per  cent. 


54  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

In  the  counting  plates  made  after  sulfiting  only  wine  yeast  was 
found  and  in  increased  numbers.  All  the  other  kinds  had  disappeared 
or  failed  to  develop.  This  shows  plainly  the  effectiveness  of  moderate 
doses  of  S02  in  paralyzing  injurious  organisms.  It  is  possible  that 
some  of  the  apiculatus  and  wild  yeast  might  have  recovered  in  time 
if  afforded  the  opportunity,  but  if  a  starter  of  good  yeast  were  used 
soon  after  sulfiting  the  fermentation  would  be  over  and  the  wine  safe 
before  they  could  develop. 

4.  effect  of  so2  in  defecation.  In  making  white  wine,  it  is  a  com- 
mon practice,  after  separating  the  must  from  the  pomace  by  draining 
and  pressing,  to  allow  it  to  clear  by  settling  and  then  to  separate  it 
from  the  sediment  by  drawing  off  into  clean  vats.  This  practice  is 
known  as  "defecation,"  and  is  possible  only  when  fermentation  fails 
to  start  within  about  forty-eight  hours.  This  delay  of  fermentation  is 
usually  insured  by  means  of  sulfuring  or  sulfiting. 

Defecation  removes  most  of  the  suspended  inert  matters  such  as 
fragments  of  the  grape,  and  soil  particles.  It  removes  also  a  part  of 
the  living  cells  of  micro-organisms.  When  sulfurous  acid  is  used  we 
obtain,  in  addition  to  this  mechanical  separation,  the  paralyzing  effect 
already  discussed.  Tests  were  undertaken  to  determine  the  relative 
part  of  each  of  these  factors  in  the  results. 

Elimination  of  Inert  Solids.  Laboratory  tests  by  R.  "W.  Bettoli* 
demonstrated  the  difficulty  of  defecation  by  means  of  refrigeration 
except  with  musts  of  exceptionally  clean  and  sound  grapes  which  need 
it  least.  Certain  molds  and  wild  yeasts  grow  at  very  low  tempera- 
tures and  produce  sufficient  fermentation  to  prevent  an  adequate 
settling  of  impurities.  Previous  pasteurization  is  even  less  effective, 
at  least  with  the  musts  used,  on  account  of  the  increase  in  the  viscosity 
of  the  liquid  due  to  changes  in  the  pectic  matters  which  prevented 
settling. 

Where  S02  was  used  at  the  rate  of  150  milligrams  per  liter  the 
defecation  was  complete,  the  supernatant  liquid  being  perfectly  clear 
to  the  eye  and  containing  no  suspended  matter  which  could  be  weighed 
after  passing  the  clear  must  through  a  gooch  filter.  The  original  must 
tested  by  weighing  a  gooch  filter  through  which  it  had  been  passed 
showed  .554  per  cent  of  suspended  floating  matter.  This  is  sufficient 
to  cause  a  bulky  sediment  in  a  wine  made  from  undefecated  must. 
The  presence  of  so  large  a  bulk  of  impurities  during  fermentation  can 
hardly  fail  to  have  an  injurious  effect  on  the  odor,  taste  and  finer 
qualities  of  the  wine. 

These  tests  give  us  no  measure  of  the  extent  to  which  elimination 
of  the  cells  of  micro-organisms  is  carried    by    defecation.      Many    of 


*Loc.  cit. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


55 


these  cells  will  pass  through  an  ordinary  filter,  and  even  if  they  were 
all  retained  their  weight  is  too  small  for  determination.  Their  elimina- 
tion can  be  determined  only  by  cultural  and  microscopical  methods. 

Bettoli*  has  shown  that  the  number  of  active  cells  in  a  must  can  be 
reduced  by  defecation  with  from  100  milligrams  to  300  milligrams  of 
S02  per  liter  from  1,240,853  per  c.c.  to  255  per  c.c.  at  22°  C.  and  to  1,500 
per  c.c.  at  28°  C.  in  forty-eight  hours.  The  efficiency  of  the  smallest 
amount  of  S02  was  very  little  inferior  in  this  respect  to  that  of  the 
larger.  The  lower  efficiency  at  the  higher  temperature  probably  repre- 
sents a  slight  mulplication  of  the  cells  which  were  not  eliminated. 

These  results  show  the  total  reduction  of  active  cells  due  both  to 
settling  and  to  the  paralyzing  effect  of  the  S02.  A  measure  of  the  part 
of  each  of  these  actors  is  given  by  comparison  with  determinations 
of  the  numbers  of  active  cells  in  the  sediments  formed.  In  the  sediments 
the  average  number  of  active  cells  was  found  to  be  615  per  c.c.  at 
22°  C.  and  7,747  per  c.c.  at  28°  C.  This  indicates  that  the  greater 
part  of  the  cells  were  rendered  inactive  by  means  of  the  S02  and  that 
only  a  relatively  small  number  were  removed  by  actual  settling.  The 
results  are  shown  in  more  detail  in  Table  20. 


TABLE  No.  20. 

Elimination  of  micro-organisms  by  defecation. 

Number  of  active  cells  originally  in  must  1,240,853  per  cubic  centimeter. 


S02  used, 
milligrams  per  liter. 

Temperature 
C. 

Number  of  active  cells  per 
cubic  centimeter. 

Elimination  per  cent. 

In  sediment. 

In  clear  juice. 

Sediment. 

Clear  juice. 

100 

22 

22 
22 
22 
28 

1,005 

645 

585 

225 

1fi.857 

45 

630 

315 

30 

3,810 

180 

1,695 

345 

99.91 
99.94 
99.95 
99.98 
98.64 
99.59 
99.51 
99.75 

99.99 

150 

99.94 

200 

99.97 

300 _ 

99.99 

100 

99.69 

150 __ 

28               5.055 

99.98 

200 

300 

28 
28 

6,045 
3,030 

99.86 
99.97 

This  table  shows  that  on  the  average  99.92  per  cent  of  the  active 
cells  were  eliminated  from  the  clear  must  and  99.56  per  cent  from  the 
sediment,  leaving  only  .08  per  cent  in  the  former  and  .44  per  cent  in 
the  latter  capable  of  immediate  development.  The  tests,  however,  do 
not  show  the  nature  of  the  cells  which  remain,  a  matter  of  importance. 

Elimination  of  Molds  and  Wild  Yeasts.  Laboratory  experiments 
with  apple  juice  showed  that  defecation  with  100  milligrams  of  S02 
per  liter  eliminated  in  twenty-four  hours  all  active  micro-organisms  in 


*Loc.  cit. 


56  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

both  the  clear  juice  and  the  sediment  except  the  true  yeasts.  All  the 
true  yeasts  were  eliminated  from  the  clear  juice  when  300  and  400 
milligrams  per  liter  were  used.  In  the  sediment  active  true  yeasts  were 
found  in  small  numbers  even  when  400  milligrams  per  liter  were  used. 

These  tests  indicate  that  the  unfavorable  activities  of  injurious 
organisms  can  be  completely  prevented  by  the  use  of  S02  alone  with- 
out defecation  by  settling.  The  utility  of  the  settling  is  simply  to 
remove  the  solid  impurities  which  would  depreciate  the  quality  of  the 
wine  if  they  remained  present  during  fermentation.  Except  perhaps 
with  very  inferior  grapes,  this  result  is  of  much  less  importance  than 
the  prevention  of  the  activities  of  injurious  organisms.  Defecation  by 
refrigeration  is  therefore  much  inferior  in  its  effects  to  defecation  by 
means  of  S02;  in  other  words,  the  sulfiting  of  the  must  is  of  more 
importance  than  the  clearing  by  settling.  When  sulfiting  alone  is 
used,  after  a  delay  of  more  or  less  extent,  depending  on  how  much  S02 
is  used,  the  must  will  finally  go  through  a  practically  pure  fermenta- 
tion ;  that  is,  the  true,  natural  wine  yeasts  present  will  take  exclusive 
possession  of  the  must. 

When  the  clear  sulfited  must,  however,  is  drawn  off  the  sediment 
before  the  start  of  fermentation  there  may  be  little  or  no  yeast  present. 
In  this  case  the  delay  of  fermentation  may  be  very  great  or,  worse  still, 
it  may  be  due  to  micro-organisms  which  get  into  the  must  after  defeca- 
tion and  these  micro-organisms  are  very  likely  in  many  cases  not  to  be 
wine  yeast  principally.  Whenever  sulfiting  and  defecation  are  both 
practised,  a  starter,  preferably  of  pure  yeast,  should  be  used  immedi- 
ately after  removal  of  the  must  from  the  defecating  vat. 

Observations  in  the  winery  corroborate  these  results  obtained  in  the 
laboratory.  A  must  made  from  Green  Hungarian  grapes  shipped  from 
the  San  Joaquin  Valley  was  defecated  in  a  1,500  gallon  vat.  The 
must  was  allowed  to  settle  after  adding  metabisulfite  at  the  rate  of 
6  ounces  to  100  gallons.  The  number  of  molds,  apiculatus  and  wine 
yeast  cells  was  determined  in  the  must  before  sulfiting  and  in  the 
cleared  must  after  settling  thirty-two  hours,  with  the  results  shown 
in  Table  21. 

TABLE  No.   21. 

Elimination  of  micro-organisms  in  a  1500-gallon  defecating  vat. 

( 
Molds.  Apiculatus.  Wine  yeast. 


Original  must  . 
Defecated  must 

Elimination    __. 


29,262  per  c.c. 
256  per  c.c. 

91.3% 


6,603,028  per  c.c. 
0  per  c.c. 

100% 


29,262  per  c.c. 
492  per  c.c. 

83.2% 


Bulletin  230] 


ENOLOGICAL  INVESTIGATIONS. 


57 


Fig.  5. 

A,  plate  from  undefecated  must,  showing  molds  ;  B,  plate  from  defecated  must,  show- 
ing yeasts. 

A  similiar  test  was  made  with  a  Palomino  must  of  the  same  origin. 
This  must  was  defecated  in  the  yeast  apparatus  with  4  ounces  of  meta- 
bisulfite  to  100  gallons. 

TABLE  No.  22. 
Elimination  of  micro-organisms  in  small  vat. 


Active  cells  per  cubic  centimeter. 


Original    must   

Defecated  8  hours 

Defecated  20  hours 

Elimination  at  8  hours. 
Elimination  at  20  hours 


Apiculatus.         Wine  yeasts. 


1,565,658 

660 

0 

99.96% 
100.00% 


529,133 
13,800 
16,280 

97.4% 
97.0% 


These  results  show  that  comparatively  small  amounts  of  S02  very 
rapidly  eliminated  all  the  active  cells  except  those  of  wine  yeast.  For 
the  fullest  effect,  desirable  in  the  preparation  of  starters,  defecation 
should  continue  for  at  least  twenty-four  hours.  This  will  insure  the 
absence  of  all  active  injurious  organisms.  There  will  probably  be  a 
slight  admixture  of  the  natural  wine  yeasts  of  the  grape  when  selected 
pure  yeasts  are  used,  but  this  contamination  is  in  practice  perfectly 
harmless  and  experience  with  the  use  of  selected  pure  yeasts  in  wineries 
shows  that  with  ordinary  precautions  it  is  reduced  to  very  little. 


58  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

5.    CHANGES  IN   THE  SUSCEPTIBILITY   OF   WINE   YEAST   TO   S02.      In   1905 

a  series  of  experiments  was  made  to  test  the  alleged  possibility  of  train- 
ing a  yeast  to  withstand  increased  amounts  of  S02.  A  must  of  20  per 
cent  Balling  and  .7  per  cent  acidity  was  divided  among  six  flasks,  to 
each  of  which  was  given  a  different  dose  of  S02.  They  were  then  all 
inoculated  with  a  pure  culture  of  wine  yeast.  The  flask  containing  the 
least  S02  commenced  to  ferment  first.  As  soon  as  this  occurred,  the 
flask  containing  the  next  higher  amount  was  inoculated  with  yeast  from 
the  fermenting  flask  and  so  on  until  the  end  of  the  series.  The  results 
are  shown  in  the  following  table : 

TABLE  No.   23. 
Exposure   of  yeast   to  increasing   doses   of   S02. 


Flask. 


S02  added, 
milligrams 
per  liter. 


Remarks. 


April  16,  all  flasks  inoculated. 

April  19,  (a)  fermenting,  inoculated  (&)  from  (a). 

April  20,  (b)   fermenting,  inoculated  (c)   from  (&). 

April  21,  (c)  fermenting,  inoculated  (d)  from  (c). 

April  23,  (d)   fermenting,  inoculated  (e)   from   (d). 

May  1,  (e)  fermenting,  inoculated  (f)  from  (e). 

May   8,    (d)    fermenting. 

May  15.     All  flasks  fermented  dry. 


Observations  of  this  kind  have  been  interpreted  as  showing  an 
increased  resistance  of  yeast  to  S02  when  gradually  accustomed  to  its 
presence.  The  yeast,  for  example,  in  flask  /  receiving  1,000  milli- 
grams of  S02  per  liter  had  not  caused  fermentation  at  the  end  of 
fifteen  days.  When  yeast  which  was  causing  fermentation  in  flask  e 
to  which  had  been  added  550  milligrams  of  S02  was  placed  in  flask  / 
fermentation  started  in  seven  days. 

The  correct  interpretation  of  these  facts  has  been  shown  by  later 
tests  to  be  this:  The  yeast  placed  in  flask  /  immediately  after  the 
addition  of  1,000  milligrams  of  S02  was  killed  or  weakened  because  a 
large  part  of  the  S02  was  in  the  free  form.  Fifteen  days  later,  when 
the  second  inoculation  of  yeast  was  made,  most  of  the  S02  had  changed 
to  the  combined  form  and  insufficient  free  S02  existed  to  prevent  fer- 
mentation. The  start  of  fermentation,  therefore,  was  not  due  to  a 
greater  resistance  of  the  yeast,  but  to  decrease  of  the  antiseptic  prop- 
erties of  the  S02. 

Other  experiments  made  at  the  same  time  pointed  to  an  actual 
weakening  of  the  yeast  by  exposure  to  S02.  To  investigate  this  point, 
a  series  of  tests  was  made  in  1911.  Three  series  of  nine  flasks  each 
were  filled  with  grape  must.    Each  flask  of  a  series  received  a  different 


Bulletin  230] 


ENOLOGICAL  INVESTIGATIONS. 


59 


amount  of  S02,  the  corresponding  flasks  of  each  series  receiving  the 
same  amount.  After  sulfiting,  each  flask  was  inoculated  with  1  per 
cent  of  the  same  vigorous  pure  yeast,  unaccustomed  to  sulfurous  acid. 
Series  III  consisting  of  flasks  a"  to  i"  was  left  without  further 
treatment  until  the  end  of  the  experiment.  Series  II  consisting  of 
flask  a'  to  *'  and  Series  I  consisting  of  flasks  a  to  i  were  left  for  five 
days  until  it  was  apparent  that  fermentation  had  started  in  all  the 
flasks  where  it  was  possible  without  new  inoculation.  In  twenty-four 
hours  the  first  five  flasks  of  all  series  had  commenced  to  ferment.  At 
the  end  of  five  days  none  of  the  last  three  had  commenced.  In  Series 
II,  flask  g'  was  then  inoculated  with  yeast  from  flask  f  of  the  same 
series,  accustomed  to  S02.  In  Series  I  flask  g  was  inoculated  afresh 
with  the  yeast  unaccustomed  to  S02.  This  process  was  continued 
until  all  the  flasks  of  Series  I  and  II  had  fermented.  Each  flask  of 
Series  II  was  inoculated  with  yeast  from  the  flask  immediately  below 
it,  while  at  the  same  time  the  corresponding  flask  of  Series  I  was  inocu- 
lated with  the  original  yeast  unaccustomed  to  S02.  The  same  variety 
of  yeast  was  used  in  all  cases. 

TABLE  No.   24. 
Comparison  of  accustomed  with  unaccustomed  yeast. 


Series  I. 

Series  II. 

Series  III. 

S02.  milligrams  per  liter. 

a 

a' 

a" 

200 

b 

b' 

b" 

250 

c 

c' 

c" 

300 

d 

d' 

d" 

350 

e 

e' 

e" 

400 

f 

V 

f" 

500 

9 

9' 

9" 

600 

h 

W 

h" 

800 

i 

V 

i" 

1000 

July  31,  6  p.  m. 
August  1,  8  a.  m. 
August     5,   5  p.  m. 


August  7,  8  a.  m. 

August  8,  8  a.  m. 

August  10,  8  a.  m. 

August  16,  8  a.  m. 

August  16,  8  a.  m. 

August  21,  8  a.  m. 


August  22,   8  a.  m. 
August  23,   8  a.  m. 


Inoculated  all  flasks  and  placed  at  29°  C. 

All  flasks  from  beginning  to  f,  f,  and  f"  fermenting. 

No  flask  started  above  the  f  row  in  any  series. 

Inoculated  g  with  unaccustomed  yeast. 

Inoculated  g'  with  yeast  from  flask  /". 

g  fermenting ;  g'  and  g"  not  started. 

g  fermenting  vigorously ;  gf  just  starting ;  g"  not  started. 

g  and  g'  fermenting  vigorously. 

Inoculated  h  with  unaccustomed  yeast. 

Inoculated  h'  with  yeast  from  g'. 

h  and  h'  fermenting  vigorously  ;  g"  not  started. 

Inoculated  i  with  unaccustomed  yeast. 

Inoculated  V  with  yeast  from  h'. 

i  fermenting ;  i"  not  started ;  V  not  started. 

i  fermenting  vigorously ;  i'  just  starting. 

g" ,  h"  and  fc"  not  started. 


60  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

This  experiment  shows  conclusively  that  exposure  to  S02  does  not 
increase  the  resistance  of  yeast,  but  on  the  contrary  makes  it  more 
susceptible.  This  is  shown  by  the  fact  that  flask  g  inoculated  with 
yeast  unaccustomed  to  S02  fermented  in  fifteen  hours,  while  the  cor- 
responding flask  g'  containing  the  same  amount  of  S02  and  inoculated 
with  a  yeast  accustomed  to  S02  did  not  start  to  ferment  until  after 
thirty-seven  hours;  and  flask  i  inoculated  with  unaccustomed  yeast 
started  to  ferment  in  twenty-four  hours,  while  V  with  accustomed  yeast 
required  forty-eight  hours. 

None  of  the  last  three  flasks  of  Series  III  started  at  all,  though  the 
free  S02  must  have  fallen  as  low  as  in  the  corresponding  members  of 
the  other  series  where  fermentation  took  place.  This  indicates  that 
the  yeast  was  killed  or  much  weakened  by  the  amount  of  S02  placed 
in  these  flasks. 

Another  series  of  experiments  was  made  in  the  attempt  to  make  a 
quantitative  determination  of  the  effect  on  yeast  of  prolonged  contact 
with  S02. 

A  flask  B  containing  100  c.c.  of  must,  to  which  100  milligrams  of 
S02  had  been  added  was  inoculated  with  1  per  cent  of  a  culture  of 
Burgundy  yeast.  A  similar  flask  A  without  S02  was  inoculated  with 
the  same  yeast.  Both  were  incubated  at  32°  C.  When  both  were  in 
full  fermentation,  a  flask  of  must  containing  100  milligrams  of  S02 
per  liter  was  inoculated  with  the  yeast  from  flask  B  grown  in  the  pres- 
ence of  100  milligrams  S02  and  a  similar  flask  was  inoculated  with 
yeast  from  flask  A  grown  in  must  free  from  S02.  The  progress  of  the 
fermentation  was  then  determined  by  periodical  weighing  of  the  flasks 
as  shown  in  Table  25.  The  same  procedure  was  followed  in  Series 
II  and  III  using  200  and  300  milligrams  of  S02  per  liter,  respectively, 
both  for  the  previous  training  of  the  yeast  and  for  the  fermentations. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


61 


TABLE  No.   25. 

Increase  of  susceptibility  of  yeast   by  exposure  to   S02. 

Series  I.      100  milligrams  SO*  per  liter.     Experiment  372a.     Weight  in  grains. 


Hours. 

Flask  A. 
untrained  yeast. 

Flask  Ji. 
trained  yeast. 

A — Loss  in 
weight. 

B — Loss  ill 
weight. 

0    

304.50 
303.89 
301.57 
299.85 
297.05 
295.30 
294.54 
294.45 

307.69 
307.10 
304.64 
302.80 
300.07 
298.50 
297.79 
297.65 

0 

.6 
2.93 
4.65 
7.45 
9.20 
9.96 
10.05 

0 

23   

.59 

52 

3.05 

70 

4.89 

92  

7.62 

119 

9.19 

138 

9  90 

168 

10.04 

Series  II.      200  milligrams  per  liter.     Experiment   372b. 


0 

17 

51 

78 

97 

127 

150 

173 

193 

211 

235 


321.05 
320.07 
319.10 
315.77 
315.24 
313.05 
311.81 
311.10 
310.75 
310.42 
310.20 


313.25 
313.10 
312.05 
309.80 
308.47 
306.25 
304.75 
303.67 
303.02 
302.57 
302.27 


0 
.15 
1.20 
3.45 

4.78 

7.00 

8.50 

9.58 

10.25 

10.68 

10.88 


Series  III.     300 

milligrams  per  liter.     Experiment  372c. 

0 

288.35 
287.83 
286.72 
284.55 
282.50 
280.47 
278.31 
277.59 
277.19 
276.55 

287.92 
287.72 
286.82 
284.82 
282.72 
280.82 
278.92 
277.85 
277.35 
276.67 

0 

.52 

1.63 

3.80 

5.85 

7.8S 

10.04 

10.76 

11.16 

11.80 

0 

22 

.2 

45            — 

1.1 

65  

3.1 

83 

5.20 

107     

7.10 

141     

9.00 

165  

182     

10.07 
10.57 

227      

11.25 

In  the  flasks  containing  100  milligrams  per  liter,  the  fermentations 
follow  almost  exactly  the  same  course,  showing  no  effect  from  the 
previous  exposure  to  S02.  In  the  flasks  containing  200  milligrams  the 
fermentation  in  the  flask  inoculated  with  "trained"  yeast  is  slower  at 
first  but  catches  up  by  a  more  rapid  fermentation  near  the  end.  In 
the  flask  containing  300  milligrams  the  fermentation  of  the  "trained" 
yeast  is  uniformly  slower  from  beginning  to  end. 

Both  yeasts  caused  a  slower  fermentation  in  the  presence  of  200 
and  300  milligrams  of  S02  than  in  that  of  100  milligrams. 

With  regard  to  the  rate  of  fermentation,  therefore,  we  have  two 
effects  due  to  S02,  both  tending  to  retard  it.  The  fermentation  is 
slower  in  musts  containing  the  larger  quantities  of  S02,  owing  to  the 
immediate  effects  of  the  S02  in  decreasing  the  activity  of  the  yeast. 
This  decrease  of  activity,  moreover,  persists,  so  that  a  yeast  which  has 
developed  in  the  presence  of  S02  is  less  active  than  before  exposure 
when  placed  in  musts  containing  large  doses  of  S02.  These  effects 
are  shown  in  diagram  6. 


62 


UNIVERSITY  OF  CALIFORNIA — EXPERIMENT  STATION. 


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Diagram  6. 
Comparison  of  fermentative  activity  of  "trained"  and  "untrained"  yeast. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


63 


The  theory  that  yeast  can  be  gradually  "trained"  to  withstand 
increasing  doses  of  S02  is  incorrect  under  the  conditions  covered  by 
these  tests.  On  the  contrary,  its  susceptibility,  as  evidenced  by  the 
rate  of  fermentation,  is  increased. 

This  decrease  in  rapidity  may  be  an  advantage  in  aiding  cool  fer- 
mentation for  as  indicated  by  these  tests  it  is  principally  at  the  start. 
In  practice  it  is  found,  however,  that  little  aid  is  to  be  obtained  in 
this  way  in  avoiding  hot  fermentations.  The  lesson  to  be  drawn  from 
these  tests  is  that  pure  yeast  and  starters  should  be  grown  in  the 
presence  of  comparatively  small  amounts  of  S02,  in  order  to  maintain 
the  vigor  and  power  of  rapid  multiplication  necessary  to  permit  them 
rapidly  to  outnumber  all  other  micro-organisms  occurring  in  the  must. 

Tests  made  with  yeast  from  a  cellar  yeast-apparatus  gave  similar 
results.  Must  showing  25°  Balling  was  placed  in  two  series  of  flasks. 
Series  A  consisted  of  flasks  a,  b,  c,  d  and  e,  which  received  100,  150, 
200,  300  and  500  milligrams  of  S02  per  liter,  respectively.  Series  B 
consisted  of  five  flasks  a2  to  e2  sulfited  in  the  same  way.  Series  A  was 
inoculated  immediately  with  a  vigorous  yeast  grown  in  must  without 
S02.  Series  B  was  inoculated  at  the  same  time  with  the  same  variety 
of  yeast  which  had  been  grown  for  a  month  in  the  yeast  apparatus  in 
must  sulfited  to  the  extent  of  200  milligrams  per  liter.  The  yeast 
in  the  apparatus  was  pure  and  apparently  vigorous.  Moreover,  it 
had  given  excellent  results  in  fermenting  wine  on  a  practical  scale. 
The  course  of  fermentation  in  the  various  flasks  was  observed  by  daily 
readings  of  the  Balling  per  cent. 

Of  the  flasks  inoculated  with  "untrained"  yeast,  3  fermented,  but 
only  one  of  the  flasks  receiving  "trained"  yeast.  The  course  of  the 
fermentation  is  shown  in  Table  26. 


TABLE  No.   26. 
Weakening  of  yeast  by  exposure  to  S02. 


Hours. 

A — Unaccustomed  yeast. 
Balling  per  cent. 

B — Accustomed 

yeast. 
Balling  per  cent. 

a 

100  milligrams. 

b 
150  milligrams. 

c 

200  milligrams. 

a2 
100  milligrams. 

0 

25.25 

25.25 

25.25 

25.25 

24.00 

20.00 

10.50 

7.00 

4.00 

.5 

0 

25.25 

25.25 

25.25 

25.25 

25.00 

21.00 

14.00 

8.50 

5.00 

1.0 

0 

25  25 

96 _. 

25.25 
25.00 
24.00 
18.00 
13.50 

6.00 

3.50 
.50 

0 

0 

25.25 

120 _ 

25.00 

145 

25.25 

168 

16  00 

193 

11.50 

221 

5  00 

240 

2.50 

268  _ 

0 

292  _ _ 

0 

315 

0 

64  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

Here,  as  in  the  former  series  of  experiments  the  accustomed  and 
the  unaccustomed  yeasts  gave  almost  identical  results  in  the  must 
containing  only  100  milligrams  S02  per  liter.  Where  larger  amounts 
were  used,  however,  the  accustomed  yeast  showed  an  increase  of  sus- 
ceptibility by  failing  to  grow  in  musts  containing  150  and  200  milli- 
grams per  liter  in  which  the  unaccustomed  yeast  grew  readily. 

In  these  experiments  the  yeast  was  added  soon  after  the  addition  of 
the  sulfite  while  a  large  amount  of  the  S02  still  existed  in  the  free 
state.  In  practice,  the  addition  of  yeast  should  be  deferred  for  at 
least  twelve  hours  after  sulfiting,  especially  where  more  than  100  milli- 
grams per  liter  of  S02  is  employed.  If  this  precaution  is  taken,  there 
need  be  no  fear  that  the  yeast  will  lack  vigor  if  it  has  been  grown  in 
the  presence  of  only  moderate  amounts  of  S02. 


III.    UTILITY  AND  METHODS  OF  APPLICATION  OF  PURE 
YEAST  IN  WINE-MAKING. 

(a)  Present  Status  of  Pure  Yeast  in  Wine-making  in  California. 
In  1893  the  station  commenced  to  investigate  the  possibility  of  improv- 
ing our  wines  by  the  use  of  pure  and  selected  yeast.  The  first  tests 
with  a  Johannisberg  wine-yeast  from  Geisenheim  on  the  Rhine  were 
made  on  a  small  scale  at  the  station  cellar.  The  results  were  promising 
and  in  1895  a  number  of  pure  yeasts  from  the  Rhine,  Medoc,  Bur- 
gundy, Sauternes,  Italy  and  Algeria  were  tested  at  the  station  and 
distributed  to  wine-makers  in  various  parts  of  the  state  for  trial. 

The  conclusions  drawn  from  the  small  station  tests  were  that  the 
use  of  pure  yeast  promoted  a  quicker  fermentation,  a  more  prompt 
clarification  and  produced  a  wine  of  cleaner  taste  and  with  some 
improvement  in  bouquet  and  flavor.  The  tests  at  wineries  were  less 
favorable.  In  many  cases,  where  the  pure  yeasts  were  used,  the  wines 
failed  to  ferment  to  dryness,  while  the  naturally  fermented  wines 
became  quite  dry.  This  was  especially  marked  in  the  case  of  the 
German  yeast  which  produced  a  dry  wine  only  in  one  case.  The  suc- 
cessful fermentation  was  that  of  a  light  Burger  must  and  the  resulting 
wine  was  much  superior  to  the  witness.  The  cause  of  the  failures  of 
this  yeast  was  its  unsuitability  to  our  heavy  musts.  The  failure  of  the 
other  yeasts  seems  to  have  been  due  to  the  fact  that  the  addition  of 
yeast  promoted  a  rapid  development  of  the  fermentation,  and  a  rise 
of  temperature  to  a  point  that  paralyzed  the  yeast  before  the  sugar 
had  all  been  eliminated. 

These  results  showed  that  a  proper  selection  of  yeast  was  necessary 
and  that  the  use  of  pure  yeast  without  some  means  of  controlling  the 
temperature  of  fermentation  was  liable  to  do  more  harm  than  good. 


Bulletin  230]  ENOLOGICAL   INVESTIGATIONS.  65 

Since  1895  the  work  of  testing  pure  yeast  and  the  methods  of  appli- 
cation have  been  continued.  Many  yeasts  from  most  of  the  chief 
wine-producing  regions  have  been  imported  and  tested  in  comparison 
with  yeasts  isolated  from  Californian  grapes  and  wines.  The  results 
have  shown  that  before  using  pure  yeast  some  means  of  controlling  the 
injurious  yeasts,  molds  and  bacteria  must  be  adopted.  This  means 
has  been  found  in  the  proper  use  of  sulfurous  acid.  With  this  help 
the  improvements  obtained  in  the  small  scale  station  tests  can  be 
realized  in  practice.  All  the  yeasts  tested,  both  imported  and  native, 
have  given  good  results  with  the  exception  of  some  German  yeasts 
which  were  unsuited  to  heavy  musts.  On  the  whole,  the  yeasts  obtained 
from  the  Champagne  and  Burgundy  districts  have  been  found  the  most 
suitable  and  have  given  the  greatest  improvement  in  increase  of  quality 
and  in  simplifying  the  handling  of  the  wine.  For  this  reason  we  have 
limited  our  supply  of  yeast  to  wineries  to  two  or  three  forms  originat- 
ing in  these  districts,  and  they  have  given  satisfactory  results  in  all 
kinds  of  wine. 

Whether  other  yeasts  will  give  better  results  in  special  cases  can  be 
determined  only  with  time  and  the  multiplication  of  experiments.  The 
improvement  obtained  by  the  use  of  these  yeasts  is  so  much  greater 
than  any  differences  existing  between  different  pure  yeasts,  that  it  seems 
safer  to  use  them  exclusively  in  our  large  scale  tests  until  the  methods 
and  advantages  of  the  use  of  pure  yeast  are  better  understood  by  most 
cellar-men. 

(6)  Organisms  found  on  Californian  Grapes.  As  pointed  out  in 
Bulletin  213,  the  spores  of  any  organism  carried  by  the  wind  may  be 
found  on  the  surfaces  of  grapes  as  they  come  to  the  winery.  Many 
of  these  organisms  are  incapable  of  growing  on  grapes  or  in  must  or 
wine.  A  large  number,  however,  are  capable  of  growing  on  the  grapes 
when  the  conditions  are  favorable  and  many  will  grow  in  the  must 
and  some  in  the  wine  itself.  They  are  all  useless  with  the  exception 
of  the  wine  yeast  and  many  of  them  highly  injurious  in  wine-making. 
The  principal  injurious  forms  have  been  described  in  Bulletin  213. 
Some  of  the  most  troublesome  often  occur  in  abundance  and  always 
in  much  larger  numbers  than  the  necessary  wine  yeast.  In  fact,  the 
latter  is  often  present  in  such  small  numbers  that  it  cannot  be  found 
by  ordinary  methods. 

The  possibility  of  restraining  the  activities  of  these  troublesome 
organisms  in  wine-making  by  the  use  of  S02  has  already  been  dis- 
cussed. Its  necessity  is  made  evident  by  investigations,  made  during 
the  past  season,  on  the  kinds  and  numbers  of  micro-organisms  occur- 
ring naturally  on  Californian  grapes  as  they  hang  in  the  vineyard 

4—230 


66  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

and  as  they  arrive  at  the  cellar  after  gathering,  hauling  and  shipping. 
The  results  emphasize  the  need  of  the  use  of  S02  and  the  utility  of 
the  employment  of  selected  yeast. 

1.  Davis.  On  a  sample  of  Muscat  grapes  gathered  in  the  vineyard 
of  the  University  Farm  at  Davis  were  found  in  large  numbers,  3  molds, 
3  wild  yeasts,  and,  in  small  numbers,  2  wine  yeasts. 

TABLE  No.  27. 
Micro-organisms*  found  on  grapes  at  Davis. 

1.  Green  mold   (Penicillium  sp.). 

2.  Gray  mold    (Mucor  sp.). 

3.  Black  mold   (Aspergillus  sp.). 

4.  Apiculate  yeast   (Saccharomyces  apiculatus). 

5.  Film- forming  wild  yeast  (Saccharomyces  sp.). 

6.  Sediment  wild  yeast    (Saccharomyces  sp.), 

7.  Wine  yeast  (2  forms)    (Saccharomyces  ellipsoideus) . 

This  is  typical  of  what  may  be  found  on  any  ripe  grapes  in  any 
vineyard  in  California  or,  in  fact,  in  any  region.  If  the  grapes  are 
gathered  carefully  while  sound,  hauled  to  the  winery  promptly  and 
properly  handled  by  the  wine  maker,  the  three  molds  would  do  no 
harm.  If  the  grapes  are  injured  by  insects,  diseases,  rough  handling 
or  rain,  if  they  are  kept  for  a  day  or  two  before  being  crushed,  these 
molds  would  very  much  depreciate  their  value  and  even  spoil  the  wine 
made  from  them. 

The  apiculate  and  other  wild  yeasts  may  render  the  wine  inferior  by 
developing  in  the  fermentation  vats  and  casks  unless  S02  is  properly 
used.  The  wine  yeasts  from  such  grapes  as  these  will  sometimes  not 
develop  at  all  or  not  until  after  the  others  have  increased  sufficiently 
to  seriously  injure  the  quality  of  the  wine.  This  is  what  often  occurs 
with  the  first  grapes  of  the  season.  As  the  season  advances,  the  wine 
yeast  gradually  increases  in  the  grape  boxes,  crushers  and  conveyers 
so  that  when  S02  is  used  to  restrain  the  injurious  yeasts,  the  natural 
wine  yeasts  ultimately  prevail.  This  method  of  obtaining  our  wine 
yeast  from  dirty  crushers  is,  however,  uncertain  and  illogical.  With 
grapes  from  some  localities,  it  usually  gives  us  finally  a  sufficiently  good 
yeast,  with  others  it  is  more  uncertain.  In  all  cases  the  kind  of  wine 
yeast  we  finally  obtain  is  liable  to  vary  from  year  to  year,  and  it  is 
much  better  practice  to  start  at  the  beginning  of  the  vintage  with  a 
pure  culture  of  a  suitable  and  tested  yeast. 

The  apiculate  yeast  found  on  the  Davis  grapes  was  tested  and  pro- 
duced a  slow  fermentation,  which  at  the  end  of  three  weeks  had  pro- 

*  These  observations  refer  only  to  such  micro-organisms  as  were  present  in  notable 
numbers,  i.  e.,  usually  hundreds  or  thousands  in  every  drop  of  must  and  which  grew 
readily  in  must. 


Bulletin  230]  ENOLOGICAL   INVESTIGATIONS.  67 

duced  only  .3  per  cent  of  alcohol.  To  do  this,  it  destroyed  2.1  per  cent 
of  sugar,  from  which  a  wine  yeast  would  have  produced  over  1  per  cent 
of  alcohol.  The  must,  after  fermentation,  had  a  fruity  odor,  and 
cleared  very  slowly.  It  thus  showed  two  of  the  chief  defects  of 
apiculatus  fermentation,  viz.,  waste  of  sugar  and  persistent  cloudiness 
of  the  wine. 

The  film-forming  yeast  gave  a  heavy  surface  growth  and  very  little 
fermentation.  The  liquid  acquired  a  disgusting  odor  and  taste  and 
lost  4.3  per  cent  of  sugar  with  the  production  of  only  .3  per  cent  of 
alcohol. 

The  second  variety  of  wild  yeast  made  a  heavy  sediment  growth  and 
caused  a  short  and  feeble  fermentation.  It  gave  the  must  at  first  a 
fruity  odor  and  after  several  weeks  a  decidedly  rancid  odor  and  taste, 
due  probably  to  the  formation  of  butyric  acid.  It  produced  only  2 
per  cent  of  alcohol. 

The  two  varieties  of  wine  yeast  gave  a  vigorous  fermentation,  fer- 
menting to  dryness  a  must  showing  28  per  cent  Balling.  They  were  of 
similar  appearance  under  the  microscope,  but  one  gave  a  very  fine- 
grained pasty  sediment,  while  the  sediment  of  the  other  was  flocculent 
and  more  bulky.  They  were  isolated  only  at  the  end  of  the  spontaneous 
fermentation  of  the  must  and  were  not  found  on  the  fresh  grapes, 
showing  that  they  were  present  in  much  smaller  numbers  than  the 
injurious  forms. 

2.  Contra  Costa  County.  The  principal  micro-organisms  of  grapes 
grown  and  crushed  in  Contra  Costa  County  were  isolated  from  the 
must  as  it  came  from  the  crusher.    The  following  forms  were  found: 

TABLE  No.  28. 
Micro-organisms  found  in  must  of  Contra  Costa  County  grapes. 

1.  Green  mold  (Penicillium  sp.) 

2.  Gray  mold   (Mucor  sp. ) 

3.  Black  mold   (Aspergillus  sp.) 

4.  Apiculate  yeast    (Saccharomyces  apiculatus). 

5.  Wild   yeast  growing   rapidly   in   must,   forming  a    thick    leathery    film    but 

causing  little  fermentation. 

6.  Wild  yeast  with  large  oblong  cells  forming  a  thin  film,  thick  sediment  and 

causing  little  fermentation. 

7.  Wine    yeast    forming    a    fine-grained    pasty    sediment   and    causing    a    good 

fermentation. 


68 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


3.  Acampo.  Grapes  grown  at  Acampo  and  shipped  to  Martinez 
showed  a  similar  choice  selection  of  forms: 

TABLE  No.  29. 
Micro-organisms  found  in  must  of  grapes  from  Acampo. 

1.  Green  mold  (Penicillium  sp.). 

2.  Gray  mold   (Mucor  sp.). 

3.  Gray  mold    (Botrytis  sp.). 

4.  Black  mold   (Aspergillus  sp.). 

5.  Apiculate  yeast   (Saccharomyces  apiculatus). 

6.  Wild  yeast — Spherical,  film-forming  \ 

7.  Wild  yeast — Large,  oblong,  film-forming  I    (Saccharomyces  sp.). 

8.  Wild  yeast — Elongated,  film-forming         ) 

9.  Wine  yeast — Small,  ellipsoidal  (Saccharomyces  ellipsoideus) . 
10.     Wine  yeast— Spherical    (Saccharomyces  ellipsoideus). 

Some  of  the  musts  examined  were  extracted  from  clean,  sound  grapes, 
others  from  grapes  in  a  more  or  less  broken  or  moldy  condition.  Prac- 
tically, the  same  kinds  of  organisms  were  found  in  all,  and  in  all  cases 
the  wine  yeasts  were  in  a  very  insignificant  minority.  The  condition 
of  the  grapes  made  little  difference  in  the  relative  number  of  wine  yeast 
cells  present,  but  influenced  considerably  the  total  number  of  all  kinds. 

To  give  a  quantitative  idea  of  the  relative  number  of  the  various 
forms  in  various  musts,  counts  were  made  in  the  must  as  it  came  from 
the  crusher.  The  method  used  was  to  count  the  number  of  colonies, 
which  developed  from  a  measured  quantity  of  must  in  plates  of  gelatin 
must. 

TABLE  No.   30. 
Number  of  micro-organisms  in  various  musts. 
Number  of  active  cells  in   1   cubic  centimeter. 


Must. 

No.  198. 

No.  199. 

No.  194. 

No.  261. 

No.  202. 

Molds    

59,000 

28,000 
0 

1,300 

1,267,000 
0 

29,000 

8,603,000 
29,000 

98,000 

7,341,000 
59,000 

1,600,000 

Wild  yeasts  (plus  apic- 
ulatus)   

2,880,000 

Wine  yeasts  

20,000 

No.  198  was  Zinfandel  grown  in  the  neighborhood  and  crushed  in 
good  condition.  It  shows  the  smallest  total  number  of  active  cells.  All 
that  developed,  however,  were  injurious  molds  and  wild  yeasts.  This 
does  not  mean  necessarily  that  no  true  wine  yeast  was  present.  It 
simply  means  that,  if  there,  it  was  in  such  small  amounts  that  the 
methods  used  failed  to  reveal  its  presence. 

No.  199  was  Alicante  Bouschet  grown  at  Acampo  and  shipped  to 
Martinez.  When  crushed  they  were  in  fairly  good  condition.  The 
number  of  mold  cells  was  very  small,  but  that  of  the  wild  yeasts  high ; 


Bulletin  230]  ENOLOGICAL  INVESTIGATIONS.  69 

owing  probably  to  development  and  multiplication  on  broken  berries  in 
transit.    The  wine  yeast  was  again  insufficiently  numerous  to  be  found. 

No.  194  was  Green  Hungarian  grown  in  San  Joaquin  County  and 
shipped  to  Martinez.  The  grapes  were  not  in  such  good  condition  as 
the  Bouschets.  Being  tender  and  juicy  they  suffered  more  in  shipment. 
Wine  yeast  was  found  in  this  must  in  moderate  numbers,  but  the  wild 
yeasts  were  in  such  large  numbers  that  the  ratio  between  them  was 
probably  little  different  from  that  in  No.  198. 

No.  202  was  second  crop  Zinfandel  in  very  moldy  condition. 

No.  261  was  Palomino  must  from  grapes  that  arrived  in  very  poor 
condition. 

These  observations  show  the  necessity  of  crushing  the  grapes  as  soon 
as  possible  after  gathering  to  prevent  the  growth  of  injurious  molds. 
The  practice  of  shipping  grapes  in  boxes  or  loose  in  cars  is  objection- 
able as  it  gives  the  molds  an  opportunity  to  develop  and  depreciate 
the  value  of  the  grapes  before  they  reach  the  winery.  A  much  better 
practice  is  to  crush  the  grapes  at  or  near  the  vineyard  into  tank  cars  in 
which  they  are  transported  to  the  winery. 

They  show  also  the  need  of  using  sulfurous  acid  as  soon  as  the  grapes 
are  crushed  in  order  to  prevent  the  development  of  the  apiculate  and 
other  injurious  yeasts  which  are  always  present.  Grapes  which  are 
crushed  and  sulfited  at  the  vineyard  may  be  transported  in  tank  cars 
in  perfect  condition  to  the  winery  even  though  they  are  two  or  three 
days  on  the  way. 

The  need  of  using  a  starter  of  good  yeast  is  also  strongly  indicated. 
This  starter  should  be  added  at  least  several  hours  after  the  sulfiting. 
When  sulfited  grapes  are  shipped  the  yeast  should  be  added  as  soon  as 
the  grapes  are  placed  in  the  fermentation  vats  in  the  winery. 

(c)  Methods  of  Transmitting  Pure  Yeasts.  The  station  has  been 
supplying  yeast  cultures  to  wine-makers  for  many  years  and  their 
utility  has  been  thoroughly  demonstrated.  It  is  very  important,  how- 
ever, that  only  a  suitable  yeast  should  be  used.  Some  of  the  yeasts, 
sent  out  at  first,  were  unsuited  to  Californian  musts  and  conditions. 
As  a  rule,  yeast  is  needed  which  is  able  to  ferment  musts  of  high  sugar 
contents  and  which  can  withstand  high  temperatures.  Certain  German 
wine-yeasts  tried  failed  in  this  respect.  Many  yeasts  have  given  good 
results,  but  the  most  generally  successful  are  those  of  Champagne  and 
Burgundy  type.  These  yeasts  are  thoroughly  suited  to  our  musts, 
and  moreover  have  the  excellent  property  of  forming  a  heavy  granular 
sediment  which  settles  rapidly  and  leaves  the  wine  bright  very  shortly 
after  fermentation. 

Occasionally  yeasts  sent  to  wineries  have  failed  to  develop.  This  is 
due  to  the  fact  that  they  soon  lose  activity  in  the  liquid  form  in  which 


70  UNIVERSITY  OP   CALIFORNIA EXPERIMENT  STATION. 

we  have  sent  them  and,  unless  used  within  a  week  from  the  time  at 
which  we  prepare  them,  they  are  difficult  to  revive  by  the  methods 
available  to  the  wine-maker. 

Such  yeasts  are  not  permanently  injured  and  when  revived  by  suit- 
able means  regain  all  their  qualities.  By  transferring  to  fresh  sterilized 
must  and  aerating  thoroughly,  they  may  in  a  few  days  be  rendered 
active  again.  If  placed  in  sterilized  must  without  aeration,  they  may 
not  develop  at  all.  If  put  directly  into  fresh  unsterilized  must  they  may 
develop  so  slowly  as  to  be  overwhelmed  by  the  wild  yeasts  present. 

When  yeasts  are  sent  out  to  the  winery  to  be  used  directly  in  fer- 
menting vats  as  they  are  received  they  should  not  be  more  than  a  few 
days  old  when  used.  When  used  first  to  start  a  yeast-propagating 
apparatus,  they  may  be  used  safely  when  they  are  several  weeks  old, 
providing  proper  precautions  are  taken  to  avoid  contamination  with 
other  micro-organisms  before  they  have  regained  their  full  activity.  It 
has  been  shown  that  yeast  grown  on  a  solid  culture  medium  retains  its 
vigor  much  longer  than  when  grown  in  a  liquid  medium.  This  is 
usually  ascribed  to  the  fuller  aeration  which  the  yeast  receives  when 
growing  on  the  surface  of  a  solid  medium  than  when  growing  sub- 
merged in  a  liquid.  It  is  well  known  that  growing  yeast  with  a  full 
supply  of  air  will  increase  its  vigor,  but  the  retention  of  this  vigor  is  in 
this  case  more  probably  due  to  the  fact  that  when  growing  on  the  sur- 
face of  its  nutrient  medium  it  is  protected  to  some  extent  from  contact 
with  its  own  products  such  as  alcohol.  Another  factor  is  possibly  that 
owing  to  the  fixed  position  of  the  yeast  and  the  solid  condition  of  the 
medium,  both  the  multiplication  of  the  yeast  and  the  exhaustion  of 
the  food  supply  are  retarded. 

Tests  were  made  to  compare  the  retention  of  vigor  by  yeast  grown 
in  each  way.  A  series  of  tubes  containing  grape  must  was  inoculated, 
half  with  yeast  C  and  half  with  yeast  B.  A  similar  series  containing 
solid  agar  must  was  inoculated,  while  warm  enough  to  be  fluid,  in  the 
same  way.  While  the  tubes  of  agar  must  were  cooling  they  were  rolled 
so  that  the  solid  nutrient  material  was  spread  in  a  thin  layer  over  the 
inner  surfaces  of  the  tube.  At  intervals  after  inoculation,  the  con- 
tents of  a  tube  of  each  lot  were  transferred  to  a  flask  of  sterile  must 
and,  two  days  after  this  transfer,  the  number  of  living  cells  in  the 
flask  was  counted.  The  numbers  thus  obtained  give  a  measure  of  the 
number  of  living  cells  in  the  original  tube  at  the  time  of  the  transfer, 
and  of  the  rapidity  with  which  they  multiplied  in  the  fresh  must. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


71 


TABLE  NO.  31. 

Relative  stability  of  liquid  and  solid  yeast  cultures. 


Age   of   culture   in   tubes. 


Yeast  C. 
Cells  per  cubic  centimeter. 


Liquid  culture.        Solid  culture 


Yeast  B. 
Cells  per  cubic  centimeter. 


Liquid  culture.        Solid  culture 


1  day  . 

2  days 

3  days 

4  days 
16  days 
32  days 


105,000,000 
65,000,000 
92,000,000 
45,000,000 
50,000,000 
4,000,000 


40,000,000 
85,000,000 
95,000,000 
65,000,000 
25,000,000 
13,000,000 


170,000,000 

105,000,000 

100,000,000 

93,000,000 

28,000,000 

7,500,000 


80,000,000 

105,000,000 

125,000,000 

110,000,000 

71,000,000 

43,000,000 


These  results  indicate  that  during  the  first  twenty-four  hours  the 
yeast  is  more  efficient  if  grown  in  liquid  medium.  For  a  week  or  per- 
haps two  there  is  little  difference,  but,  at  the  end  of  a  month,  the  advan- 
tage is  very  plainly  with  the  yeast  on  solid  culture  medium. 

Utilizing  this  property  of  solid  cultures,  a  company  in  France  pre- 
pares a  form  of  yeast  for  wineries  under  the  proprietary  name  of 
1 '  Gelolevures. ' '  The  yeast  is  grown  on  a  thin  layer  of  nutrient  gelatine 
spread  on  a  piece  of  thin  cloth.  Several  of  these  pieces  are  suspended 
on  a  support  inside  a  sealed  tin  case.  The  directions  accompanying 
these  tins  are  to  remove  the  stopper,  fill  with  sterilized  must  and  place 
in  a  warm  place.  As  soon  as  fermentation  commences,  the  cover  is 
removed  and  the  whole  contents  of  the  tin  are  poured  into  a  fermenting 
vat  just  filled  with  grapes.  Tins  of  various  sizes  are  supplied  for  vats 
of  various  capacities.  The  claim  made  for  these  yeasts  is  that  they 
retain  their  vigor  indefinitely  and  obviate  the  necessity  of  preparing  a 
starter. 

Sample  tins  of  ''Gelolevures"  were  received  at  the  station  for  exam- 
ination. To  test  their  vigor,  one  of  the  tins,  three  months  old,  was 
treated  according  to  the  directions.  At  the  same  time  a  large  flask, 
containing  a  liquid  culture  of  yeast  two  months  old,  was  used  for  com- 
parison. The  liquid  in  the  latter  was  poured  off  the  yeast  sediment 
and  the  flask  filled  with  the  same  must  used  to  fill  the  tin  of  "Gelo- 
levures." The  must  in  the  tin  started  to  ferment  sooner  than  that  in 
the  flask  and  finished  fermenting  in  a  shorter  time. 

The  promptness  with  which  the  fermentation  started  in  the  "Gelo- 
levures" demonstrated  the  superiority  of  this  method  of  preparing 
yeast  where  it  is  to  be  used  directly  in  the  vats  without  the  inter- 
mediate preparation  of  a  starter,  and  where  there  is  any  considerable 
interval  between  the  preparation  of  the  yeast  and  its  use. 

A  similar  test  was  made  with  yeasts  prepared  in  the  laboratory. 
Two  small  flasks,  in  which  a  Champagne  yeast  was  grown  on  a  solid 
medium,  were  kept  in  the  laboratory  for  five  months.     At  the  end  of 


72  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

that  time,  sterile  must  was  added  to  the  flasks,  and  was  in  full  fermenta- 
tion within  twenty  hours.  Liquid  cultures  of  the  same  yeast  kept  for 
the  same  length  of  time  require  careful  manipulation  to  revive  them. 
The  reviving  of  a  weak  culture  cannot  safely  be  undertaken  at  the 
winery,  owing  to  the  great  chance  of  contamination  from  outside 
organisms.  A  strong,  solid  culture,  on  the  other  hand,  such  as  those 
contained  in  these  flasks  or  the  * '  Gelolevures, "  could  easily  be  used 
to  make  a  starter  by  ordinary  methods  easily  employed  by  a  competent 
wine-maker. 

These  tests  all  indicate  the  superiority  of  solid  cultures  for  trans- 
mitting yeast  for  use  in  wineries,  and  most  of  the  cultures  sent  out  by 
the  station  during  the  last  vintage  were  in  this  form. 

(d)  Methods  of  Use  in  Wineries.  1.  direct  application.  There 
are  two  general  methods  of  using  pure  yeast  in  wineries.  The  simplest 
in  theory  is  to  obtain  from  a  reliable  pure  yeast  laboratory  a  culture 
of  yeast  for  each  vat  of  grapes  or  cask  of  must  to  be  fermented.  This 
method,  however,  is  expensive,  involving  the  buying  and  transportation 
of  large  quantities  of  yeast. 

It  has  been  found,  empirically,  that  it  requires  about  1  per  cent  of 
yeast  to  properly  start  a  vat  of  sulfited  grapes  or  defecated  must.  This 
means  that  ten  gallons  of  a  vigorous  culture  of  yeast  should  be  added 
to  1,000  gallons  of  must  to  ensure  that  the  fermentation  is  due  prin- 
cipally to  the  yeast  added. 

Determinations  of  the  numbers  of  cells  present  in  yeast  cultures,  and 
in  sulfited  musts  at  the  winery  indicate  that  these  proportions  are  suit- 
able. An  average  vigorous  culture  of  wine-yeast  in  grape  must  will 
contain  about  200,000,000  active  cells  per  cubic  centimeter.  A  properly 
sulfited  must  will  contain  no  injurious  wild  yeast,  and  the  natural  wine 
yeast  will  be  reduced  to  a  few  tens  of  thousands  per  cubic  centimeter, 
even  in  musts  made  from  grapes  in  bad  condition.  A  1  per  cent  addi- 
tion of  a  vigorous  culture  of  pure  yeast  would  introduce  approximately 
2,000,000  cells  per  cubic  centimeter  into  the  whole  mass.  The  pure 
yeast  cells,  therefore,  would  be,  even  in  unfavorable  cases,  a  hundred 
times  more  numerous  than  the  natural  yeast  cells,  while,  in  ordinary 
musts  made  from  clean  grapes,  promptly  crushed  and  sulfited,  a  1  per 
cent  addition  of  pure  yeast  would  cause  the  added  yeast  to  outnumber 
the  natural  yeasts  many  thousand  times,  and  insure  a  fermentation 
which  would  be  practically  pure  so  far  as  contamination  with  other 
yeasts  is  concerned. 

A  smaller  addition  than  1  per  cent,  however,  is  not  found  advisable 
in  practice,  except  in  very  hot  weather,  when  it  may  be  reduced  a  little. 
If  too  little  is  used,  the  start  of  fermentation  is  unduly  prolonged,  and 


Bulletin  230]  ENOLOGICAL  INVESTIGATIONS.  73 

more  than  seems  theoretically  necessary  must  be  used  as  a  safety  factor 
to  provide  against  possible  unusual  contamination  of  the  grapes  or 
lack  of  vigor  of  the  culture. 

To  start  the  fermentation  of  a  vat,  containing  1,000  gallons  of 
crushed  grapes  by  adding  a  liquid  yeast  culture  directly,  would  require 
10  gallons,  which,  with  the  glass  containers,  would  weigh  approxi- 
mately 150  pounds.  If  solid  cultures  were  used,  prepared  in  glass 
flasks  or  bottles,  such  as  were  sent  out  by  the  station  during  the  last 
vintage,  the  weight  could  be  reduced  to  about  50  pounds.  The  tin 
cases  of  the  ' '  Gelolevures "  make  it  possible  to  still  further  reduce  this 
weight  to  about  15  pounds.  Even  the  last  weight  is  considerable,  and 
the  amount  of  yeast  necessary  makes  the  method  very  expensive. 

2.  preparation  of  a  starter.  Several  years  of  experience  have 
thoroughly  demonstrated  that  it  is  perfectly  practicable  for  any  intel- 
ligent cellar-man  to  commence  with  a  small  culture  of  pure  yeast,  and 
prepare  his  own  yeast  from  this  in  the  form  of  a  starter  in  any  quan- 
tity needed. 

The  method  is  perfectly  simple  and  equally  adapted  to  large  or  small 
cellars.  Expensive  equipment  in  the  form  of  pure  yeast  propagators, 
such  as  are  used  in  breweries,  is  unnecessary  and,  in  fact,  dangerous 
in  untrained  hands.  All  that  is  needed  is  an  outfit  of  tubs,  vats  and 
casks,  such  as  are  found  in  every  cellar.  These  will  vary  in  number 
and  size,  according  to  the  scale  on  which  wine  is  made  and  the  arrange- 
ment of  the  cellar.  The  yeast  apparatus,  which  was  used  in  our  winery 
experiments  during  the  last  vintage,  and  which  is  described  later,  illus- 
trates the  principles  of  the  method,  and  will  be  found  perfectly  prac- 
tical for  a  small  winery.  Modifications  to  facilitate  handling  and  to 
adapt  it  to  larger  operations  can  easily  be  devised. 

(e)  Rejuvenation  and  Increase  of  the  Pure  Culture.  When  the 
culture  of  pure  yeast  reaches  the  winery,  it  should  be  as  fresh  and 
vigorous  as  possible.  The  cellar-man  must  first  determine  whether  it 
has  the  necessary  vigor  and  rejuvenate  it,  if  necessary. 

The  next  step  is  to  increase  the  amount  of  pure  yeast  until  it  is 
sufficient  to  inoculate  the  first  starting  tub  or  vat.  The  quantity  neces- 
sary will  depend  on  the  size  of  the  apparatus.  Up  to  this  point  special 
precautions  should  be  taken  to  avoid  contamination  of  the  culture, 
and  to  insure  that  the  yeast  used  to  inoculate  the  starting  tub  is  quite 
pure. 

The  final  step  is  the  increase  of  the  yeast  in  the  starting  tub  until 
it  is  sufficient  to  start  the  first  fermentations.  The  starting  tub  or 
tubs  should  be  large  enough  to  produce  each  day  all  the  yeast  necessary 
to  start  all  the  grapes  crushed  in  one  day.    A  new  tub,  or  series  of  tubs, 


74  UNIVERSITY  OP  CALIFORNIA EXPERIMENT  STATION. 

therefore,  must  be  prepared  each  day  during  the  vintage.  The  rejuve- 
nation and  preliminary  increase,  however,  are  needed  only  for  the 
first.  All  subsequent  tubs  are  inoculated  from  the  tub  of  the  previous 
day.    Details  of  the  various  operations  are  given  below. 

(/)   Directions  for  propagating  yeast  in  wineries. 

Step  I.  Rejuvenation.  (1)  solid  cultures.  A  culture  of  pure 
yeast  is  received  from  the  station.  This  culture  is  contained  in  a 
quart  bottle,  the  neck  of  which  is  closed  with  a  tight  plug  of  cotton. 
The  interior  of  the  bottle  is  coated  with  a  layer  of  solid  culture  medium, 
consisting  of  agar-agar  and  grape  must.  In  this  medium  and  on  its 
surface  the  yeast  is  growing. 

1.  Obtain  one  quart  of  must  from  clean,  sound  grapes  not  overripe 
and  place  in  a  one  quart  Mason  preserving  jar,  which,  with  its  cover, 
should  be  thoroughly  sterilized  with  boiling  water  before  use.  The 
jar  should  not  be  filled  to  nearer  than  one  inch  from  the  top. 

2.  Place  the  jar  of  must  in  a  pot  containing  enough  water  to  reach 
about  half  way  up  the  side  of  the  jar  and  deep  enough,  so  that  it  can 
be  covered  when  containing  the  jar.  Place  its  cover  loosely  on  the 
jar,  but  do  not  screw  down. 

3.  Cover  the  pot  and  place  on  stove  until  the  water  boils.  Continue 
boiling  for  ten  to  fifteen  minutes. 

4.  Take  the  pot  from  the  stove  without  removing  the  cover  and 
allow  to  cool  nearly  to  the  temperature  of  the  room. 

5.  Moisten  the  cotton  plug  and  the  neck  of  the  bottle  containing 
the  yeast  culture  with  a  little  alcohol.  Apply  a  lighted  match.  This 
will  destroy  any  spores  which  have  settled  on  the  neck  of  the  bottle. 

6.  Remove  the  cotton  plug  with  a  pair  of  pincers,  previously  ster- 
ilized by  dipping  in  alcohol  and  flaming. 

7.  Pour  the  sterilized  and  cooled  must  from  the  Mason  jar  into  the 
opened  bottle  containing  culture.  This  is  best  done  by  means  of  a 
glass  or  metal  funnel,  previously  sterilized  by  dipping  in  boiling  water. 
Avoid  all  contact  of  the  fingers  or  other  unsterilized  surfaces  with  the 
neck  of  the  bottle,  the  inside  of  the  funnel  or  with  the  must.  The  bottle 
should  be  filled  about  four  fifths  full.  Care  should  be  taken  not  to  wet 
the  inside  of  the  neck  of  the  bottle  with  the  must. 

8.  Pass  the  cotton  plug  quickly  through  the  flame  of  an  alcohol  lamp 
and  replace  in  the  neck  of  the  bottle. 

9.  Place  the  bottle  in  a  warm  place  until  fermentation  starts.  The 
temperature  of  the  place  should  be  as  near  80°  F.  as  practicable.  If 
it  is  below  65°  F.  fermentation  will  be  slow  in  starting;  if  much  above 
90°  F.  the  yeast  may  be  injured.  A  fireless  cooker,  containing  one  or 
two  bottles  of  water  warmed  to  95°  F.,  makes  a  good  incubator.     A 


Bulletin  230]  ENOLOGICAL  INVESTIGATIONS.  75 

bucket  or  box,  lined  with  wood  wool  ("excelsior"),  in  which  the  yeast 
bottle  and  two  bottles  of  warm  water  are  buried,  and  the  whole  covered 
with  a  blanket,  is  also  suitable. 

A  culture  treated  in  this  way  should  be  in  full  fermentation  within 
twenty-four  hours. 

(2)  liquid  cultures.  The  manipulation  of  a  liquid  culture  is  sim- 
ilar, but  requires  some  special  precautions,  owing  to  the  possibility 
that  the  yeast  is  somewhat  enfeebled  by  prolonged  immersion  in  the 
liquid.  The  numbers  of  the  paragraphs  following  correspond  to  those 
of  the  paragraphs  above. 

1.  The  quart  of  must  should  be  placed  in  a  two  quart  Mason  jar, 
filling  it  not  more  than  half  full.  A  half  gallon  demijohn,  plugged 
with  cotton  wool,  and  with  the  wicker  cover  removed,  is  even  better. 

2.  Pour  the  contents  of  the  bottle  of  pure  yeast  into  the  sterilized 
and  cooled  must  in  the  two  quart  Mason  jar  and  screw  down  the  top 
of  the  latter.  If  a  demijohn  is  used,  replace  the  cotton  with  a  scalded 
cork.  Shake  the  mixture  thoroughly  for  five  minutes  to  aerate  it. 
Loosen  the  top  of  the  jar,  or  replace  the  cotton  plug  of  the  demijohn. 
This  aeration  is  very  necessary  if  the  liquid  culture  is  at  all  old,  and 
may  have  to  be  repeated  several  times. 

Step  2.  Increase.  The  first  step  in  the  process  has  provided  us 
with  1  quart  of  young  vigorous  yeast.  This  may  be  used  directly  to 
inoculate  the  first  starting  tub,  but  it  is  safer  to  increase  it  a  little 
first  unless  we  are  working  on  a  very  small  scale.  At  least  5  per  cent 
of  pure  yeast  should  be  used  to  inoculate  the  starting  tub.  The  fol- 
lowing directions  are  for  the  case  where  the  starting  tubs  are  intended 
to  supply  25  gallons  of  starter  per  day,  which  is  sufficient  for  2,500 
gallons  of  fermenting  grapes;  that  is,  the  contents  of  one  medium- 
sized  fermenting  vat.  The  directions  can  easily  be  modified  for  larger 
quantities. 

1.  Place  1J  gallons  of  clean,  fresh  grape  must  of  about  20  per  cent 
Balling  in  a  two  gallon  demijohn,  from  which  the  wicker  cover  has 
been  removed,  and  which  has  been  sterilized  with  boiling  water,  and 
plugged  with  a  tight  roll  of  absorbent  cotton. 

2.  Heat  demijohn  of  must  to  boiling  for  fifteen  minutes  in  a  covered 
boiler,  containing  a  few  inches  of  water. 

3.  Remove  from  fire  and  allow  to  cool. 

4.  Remove  cotton  plug  carefully  and  pour  the  quart  of  rejuvenated 
yeast  into  the  demijohn  of  must,  using  the  same  precautions  to  pre- 
vent contamination  explained  in  Step  1. 

5.  Aerate  the  must  in  the  demijohn  by  shaking  three  or  four  times  at 


76  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

intervals  of  three  to  four  hours,  and  leave  in  a  warm  place  until  vigorous 
fermentation  occurs. 

Step  3.  Inoculation  of  Starting  Tub.  We  now  have  L|  gallons  of 
pure  yeast,  which  is  sufficient  to  inoculate  30  gallons  of  must  in  a  start- 
ing tub. 

1.  About  twenty-four  hours  before  the  end  of  Step  2  place  about 
40  gallons  of  must  in  a  suitable  vat  or  barrel.  Add  2  ounces  of  potas- 
sium metabisulfite  (5  ounces  per  100  gallons,  equivalent  to  about  175 
milligrams  of  S02  per  liter),  and  allow  to  settle  for  about  twenty-four 
hours.  If  the  must  is  cold  and  made  from  clean,  fresh  grapes,  the 
amount  of  sulfite  may  be  reduced  one  third  to  one  half  with  advantage. 

2.  Draw  off  30  gallons  of  this  clear,  defecated  must  in  to  the  start- 
ing tub  and  warm  to  90°  F.  with  boiling  water.  If  the  must  is  very 
sweet,  the  boiling  water  may  be  added  to  it  directly.  Six  gallons  of 
boiling  water  will  raise  24  gallons  of  must  from  60°  F.  to  90°  F.  If 
the  must  has  24  per  cent  Balling,  the  water  will  reduce  it  to  about  20 
per  cent  Balling.  If  the  must  has  no  excess  of  sugar,  it  may  be  warmed 
by  floating  in  it  a  metal  bucket  or  tub  of  boiling  water,  or  by  boiling 
5  or  6  gallons  of  the  must. 

3.  Add  the  lj  gallons  of  yeast  obtained  by  Step  2  to  the  warmed 
must  in  the  starting  tub. 

4.  Aerate  thoroughly  for  ten  minutes  by  dipping  out  bucketfuls  of 
must  and  pouring  back. 

5.  Cover  tub  with  a  canvas  sheet  to  keep  out  dust  and  maintain  the 
temperature.     Aerate  and  warm  two  or  three  times,  if  necessary. 

6.  When  the  must  in  the  starting  tub  is  in  full  fermentation,  it  is 
ready  to  use  for  starting  the  regular  fermentations. 

(g)  Use  of  the  Starter.  This  preparation,  which  may  seem  some- 
what complicated  on  paper,  is  really  very  simple  when  understood, 
and,  moreover,  is  necessary  only  once.  It  should  be  done  before  the 
regular  crushing  of  grapes  commences,  and  the  first  tub  of  starter 
should  be  ready  for  the  first  load  of  grapes. 

During  the  vintage,  all  that  is  necessary  is  to  put  one  gallon  of  fer- 
menting must  from  the  starting  tub  into  every  one  hundred  gallons  of 
crushed  grapes.  At  the  end  of  the  day  the  fermenting  must  taken 
from  the  tub  is  replaced  with  fresh  defecated  must,  and  so  on  until 
the  end  of  the  vintage. 

At  least  10  per  cent  of  the  fermenting  must  should  be  left  in  the 
tub  to  start  the  new  must  added.  If  this  is  done,  the  starter  remains 
practically  pure  until  the  end  of  the  vintage.  If  the  vintage  is  long, 
it  might  be  advisable  to  add  a  fresh  supply  of  pure  yeast,  developed 
from  a  new  pure  culture,  to  the  starting  tub  about  the  middle  of  the 
vintage. 


Bulletin  230]  ENOLOGICAL   INVESTIGATIONS.  77 

Where  the  yeast  is  needed  only  once  a  day,  a  single  starting  tub  is 
all  that  is  necessary;  the  size  of  the  tub  depending  on  the  amount  of 
yeast  needed.  Where  the  yeast  is  needed  all  day,  it  is  necessary  to 
have  two  tubs  to  be  used  on  alternate  days,  as  the  must  added  at  night 
will  not  be  ready  to  use  the  next  morning. 

(//)  Stage  of  Maximum  Efficiency  of  a  Starter.  When  we  make  a 
new  addition  of  must  to  a  starting  tub,  we  dilute  the  yeast,  so  that 
there  is  a  comparatively  small  number  present  in  a  given  volume.  The 
yeast  immediately  commences  to  multiply  and  finally  reaches  a  maxi- 
mum. It  remains  at  this  maximum  number  for  some  time,  but  the 
yeast  cells  gradually  diminish  in  vigor. 

If  we  use  the  starter  too  soon,  therefore,  we  fail  to  obtain  sufficient 
yeast  cells  to  give  us  the  full  effect;  if  we  use  it  too  late,  the  yeast 
cells  have  lost  some  of  their  vigor,  they  are  less  efficient  in  overcoming 
the  competing  wild  organisms,  and  the  fermentation  is  slower. 

There  is  theoretically  some  point  between  the  commencement  of  fer- 
mentation and  the  end,  at  which  the  yeast  in  the  starter  has  its  maxi- 
mum effectiveness.  An  experiment  was  undertaken  to  determine  this 
point  of  highest  efficiency  and  to  discover  some  simple  means  of  detect- 
ing it. 

It  is  possible  to  determine  the  number  of  yeast  cells  present  by  means 
of  a  microscopical  counting  apparatus,  but  this  gives  us  no  measure  of 
the  vitality  or  degree  of  vigor  of  these  cells.  Some  of  the  cells  may  be 
young  and  vigorous,  some  old  and  decrepit,  some  dead.  By  means  of 
cultures  on  gelatine  plates,  a  better  estimate  of  the  numbers  of  vigor- 
ous cells  could  be  made,  but  this  is  troublesome  and  requires  several 
days.  Neither  method  is  suitable  for  cellar  use  in  the  press  of  the 
vintage. 

The  age  of  the  starter  gives  us  a  very  uncertain  measure.  The  con- 
dition of  the  yeast  at  the  end  of  a  certain  number  of  hours  will 
depend  on  many  conditions,  principally  the  temperature,  the  num- 
ber and  activity  of  the  cells  at  the  beginning,  and  the  amount  of 
aeration. 

A  simple  guide  was  found  in  the  disappearance  of  the  sugar.  As  the 
development  of  the  yeast  progresses,  the  Balling  per  cent  decreases. 
A  certain  decrease  of  Balling  per  cent  indicates  a  certain  amount  of 
work  done  by  the  yeast,  and  is  a  measure  of  the  number  and  vigor 
of  the  cells  present.  It  remained  to  determine  at  what  stage  in  the 
decrease  of  the  Balling  per  cent  the  yeast  possessed  its  highest  efficiency 
as  a  starter.    The  following  tests  throw  light  on  this  matter. 

A  flask  (A)  containing  must  of  22.38  Balling  and  .75  per  cent  acidity 
was  inoculated  with  a  non-agglomerating  wine  yeast.  The  culture  in 
this  flask  was  used  as  a  starter  and  a  series  of  flasks,  B  to  L,  containing 


78 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


the  same  must  was  inoculated  each  at  different  ages  of  the  starter. 
The  Balling  per  cent  of  the  starter  (flask  A)  was  determined  at  each 
inoculation  and  the  number  of  yeast  cells  present.  By  observing  the 
course  of  fermentation  in  the  various  flasks  we  are  able  to  determine 
what  Balling  degree  of  the  must  in  A  corresponds  to  the  maximum 
efficiency  of  the  yeast  when  used  as  a  starter. 

TABLE  No.  32. 


Correspondence  of  Balling 
In  Flask  A   (used  as 

per  cent  and 
a  starter  for 

number  of  yeast  cells. 
flasks  B  to  L). 

Hours. 

Balling 
per  cent. 

Yeast  cells  per 
cubic  centimeter. 

1  per  cent  of  starter 
added  to 

0  

22.38 
20.80 
18.69 
13.10 
11.31 

7.58 

4.50 
13 

0 

0 

0 

0 

18   

5,620,000 
74,385,000 
122,322,000 
166,540,000 
175,631,000 
187,926,000 
188,462,000 
190,000,000 
191,000,000 
189,000,000 
188,000,000 

Flask  B 

29   

Flask  O 

43.5___ 

Flask  D 

51.5 

Flask  E 

66.5 

Flask  F 

74.5 

Flask  G 

80.5 

Flask  H 

88.5 

Flask  I 

105.5 

Flask  J 

113.5 

Flask  K 

124.5 

Flask  L 

Under  the  conditions  of  the  experiments  the  number  of  yeast  cells 
increased  very  rapidly  until  about  half  of  the  sugar  had  disappeared. 
After  this  it  increased  slowly  until  all  the  sugar  was  gone.  Later  it 
remained  practically  stationary  until  the  end  of  the  test.  The  com- 
parative effectiveness  of  the  yeast  as  a  starter  at  the  various  stages  is 
shown  by  the  records  of  the  other  flasks. 


TABLE  No.   33. 
Fermentation  with  starters  of  various  Balling  per  cents  and  ages. 


Flask  B.     Balling 

per  cent  of  starter  20.8. 

Flask  C.     Balling 

per  cent  of  starter  18.69. 

Hours. 

Balling. 

Cells 
per  cubic 
centimeter. 

Hours. 

Balling. 

Cells 
per  cubic 
centimeter. 

0     

22.38 

21.7 

19.73 

16.33 

13.26 

9.75 

6.7 

3.62 

1.03 

0 

0 

22.38 

21.83 

19.10 

15.50 

11.91 

8.53 

6.46 

3.20 

1.03 

0 

10.5 

6,612,000 
87,609,000 
107,852,500 
173,565,000 
181,800,000 
181,830,000 

14.5 

18,678,900 

25.0  

22.5   

27,357,150 

33.0  _               

37.5 

144,635,700 

48 

44.5 

150,500,000 

56 

59.5 

178,060,750 

72 

67.5     

180,134,000 

80      

84.5  

181,830,000 

97 

185,951,500 

93.5  

106 

108.5  _. 

183,635,000 

Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


79 


TABLE  No.  33 — Continued. 
Fermentation  with  starters  of  various  Balling  per  cents  and  ages. 


Flask  D.     Balling  per  cent  of  starter  13.1. 


Flask 


Balling  per  cent  of  starter  11.31. 


Balling. 


Cells 

per  cubic 

centimeter. 


Hours. 


Balling. 


Cells 

per  cubic 

centimeter. 


0 

8 
23 

;u 

47 
55 

72 
81 

95 


22.38 

21.83 

20.36 

16.83 

12.95 

10.35 

6.20 

3.87 

0 


940,000 

4,264,740 

89,675,250 

95,047,500 

168,606,000 

172,324,000 

178,937,250 


181,576,300 


0 
15 

23 
39 

47 
63 

72 
80 
94 


22.38 

21.59 

20.26 

15.55 

15.03 

8.27 

5.94 

2.07 

0 


13,214,000 
64,053,750 
109,093,000 
136,372,500 
144,637,500 
156,627,300 
157,035,000 
165,300,000 


Flask  F.     Balling  per  cent  of  starter  7.58. 


Flask  G.     Balling  per  cent  of  starter  4.5. 


22.38 

21.57 

18.16 

16.6 

11.91 

8.79 

4.65 

1.55 

0 


144,634 

3,925,875 

55,615,250 

88,435,500 

120,975,000 

133,066,500 

140,505,000 

150,325,000 

169,432,500 


0  22.38 

17  22.36 

25  20.00 

42  14.77 

51 12.17 

65  i  7.63 

73  4.65 

89  .1 

101  —0 


15, 

6o; 

92! 
100 
103 
133 
180 
183 


124,950 
334,500 
154,750 
923,750 
312,500 
066,500 
,177,000 
,483,000 


Flask  H.     Balling  per  cent  of  starter  .13. 


Flask  I.     Balling  per  cent  of  starter  0. 


0     

22.38 

21.83 

20.00 

16.86 

12.43 

8.79 

4.78 

2.3 

0 

0     _„ _ 

22.38 

22.09 

19.73 

16.1 

11.91 

8.27 

7.24 

2.3 

0 

8     

2,727,400 
57,615,250 
78,517,500 
107,445,000 
138,851,000 
140,505,000 
152,076,000 
175,218,000 

17      

14,050,500 

25     

26     

56,202,000 

33     

40      

78,517,500 

47      

49     

99,180,000 

55     

65     

115,710,000 

71      

77      

134,719,500 

83     

95     

135,546,000 

101      

113 

140,505,000 

Flask  J.      Inoculat 
re 

ed  18  hours  a 
iched  0. 

fter  starter 

Flask  K.      Inoculat 
reached 

ed   26   hours  < 
0°   Balling. 

ifter  starter 

0     

22.38 

22.09 

17.7 

17,38 

13.47 

10.87 

6.72 

2.32 

0 

0       

22.38 

21.31 

20.78 

19.34 

13.21 

8.27 

3.6 

0 

33     

5,372,250 

28,927,500 

55,788,750 

95,874,000 

113,230,500 

129,760,500 

144,464.000 

154,555,500 

8       

7,438,500 

47      

•  24        _____ 

32,233,500 

55     

i  32      

85,129,500 

71      

54      

96,600,500 

83      

72 

117,189,500 

98      

95 

142,984,500 

116 

125 

152,902,500 

139     

Flask   L. 


Starter  used    36   hours   after   fermentation 
was  over  in  starting  flask. 


0 
8 

28 
46 
69 
99 
117 


5,785,500 
64,467,000 
108,271,500 
133,066,500 
159,514,500 
158,614,300 


80  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

Diagram   7. 

Influence   of  stage   of  starter    (indicated   by  Balling    °)    on   subsequent   fermentation. 

21  19  13  11  8  5  .1  0  0  0  0 

6  74         122         167         176         188         188         190         191         189         188 

II lltilllll 

189        /6-f-       ld%       16b       16  9      165      175       14-1       155      /53      /59 
M/1X/MUM  NUMBER   OF  ^ ERST  CELLS    IN  MILLIONS   REHCC. 

I  I  III  I  1 


H 


/oe      IOZ      95       95      9^f     tOO      101       IIZ        IIS       IZ5 
DURRVONOF  FERMENTATION  /N  HOURS. 

Stage  of  starter  (A)  expressed  by  Balling  °  and  millions  of  yeast  cells  per  cubic 
centimeter   (see  Table  No.  32). 

The  data  of  Tables  32  and  33  are  brought  together  in  Diagram  7.  The 
fermentation  in  flasks  D,  E  and  F  in  which  the  starter  was  used  when 
the  Balling  per  cent  had  fallen  to  13,  11  and  8  per  cent  respectively, 
was  the  most  rapid.  In  flasks  B  and  C  in  which  the  starter  was  used 
soon  after  commencing  to  ferment  the  fermentation  required  about 
twelve  hours  more.  This  was  probably  due  to  the  fact  that  the  starter 
contained  less  yeast  and  more  time  was  required  for  the  yeast  to 
increase  sufficiently  to  finish  the  fermentation.  In  flasks  I,  J,  K  and 
L  in  which  the  starter  was  used  after  it  had  ceased  to  ferment  the 
fermentation  was  still  slower,  requiring  about  twenty-four  hours  more. 
This  is  undoubtedly  due  to  a  weakness  of  the  activity  of  the  yeast  as  the 
number  of  yeast  cells  in  the  starter  was  at  its  maximum  when  used. 

These  results  indicate  that  the  starter  reaches  condition  of  maximum 
efficiency  about  the  time  that  the  Balling  per  cent  has  fallen  to  10  per 
cent.  This  would  probably  vary  with  the  Balling  per  cent  of  the  orig- 
inal must  used  in  making  the  starter  and  with  other  conditions,  but 
as  the  conditions  were  in  most  respects  similar  to  those  under  which  a 
starter  would  be  prepared  in  a  winery,  they  justify  the  advice  given  to 
use  the  starter  when  about  half  of  the  sugar  has  disappeared. 

As  the  starter  must  be  used  for  several  hours  during  the  day,  it 
would  be  impracticable  to  use  it  always  at  the  point  of  maximum 
efficiency,  and  this  is  not  necessary  as  it  retains  very  nearly  the 
maximum  for  some  time.  A  sufficient  rule  for  practice  is  to  wait  until 
10  per  cent  of  the  Balling  has  disappeared  and  then  use  it  as  long  as 
some  sugar  still  remains. 


Bulletin  230]  ENOLOG-ICAL   INVESTIGATIONS.  SI 

The  time  required  after  the  addition  of  new  must  to  bring  a  starter 
to  this  condition  depends  principally  on  the  amount  of  yeast  left  in 
the  starting1  tub  and  on  the  temperature  of  the  cellar. 

If  the  starter  develops  too  slowly,  it  can  be  accelerated  by  leaving  a 
larger  amount  of  yeast.  This  will  usually  control  the  time  perfectly, 
except  in  the  case  where  very  small  yeast  tubs  arc  used  or  in  very 
cold  weather,  in  which  cases  it  may  be  necessary  to  warm  the  liquid. 
Too  rapid  development  of  the  starter  is  to  be  controlled  by  the  opposite 
measures.  Where  very  large  yeast  vats  are  used,  a  cooling  coil  may  be 
necessary. 

Winery  tests  with  the  simple  yeast  apparatus  described  later  showed 
that  when  a  new  addition  of  must  was  added  to  the  yeast  tub  at  10 
a.  m.  the  yeast  was  ready  for  use  by  6  a.  m.  the  next  morning.  It  is 
necessary,  therefore,  to  have  only  one  yeast  tub  if  but  a  limited  number 
of  fermentations  are  to  be  started.  If  fermentations  are  to  be  started 
all  day,  it  would  be  necessary  to  have  two  tubs— one  for  the  morning 
and  one  for  the  afternoon. 

The  morning  tub  could  be  filled  at  about  10  a.  m.  and  would  be  ready 
to  use  from  6  a.  m.  to  10  a.  m.  of  the  following  day,  and  could  be  filled 
immediately  with  fresh  must  for  the  next  day.  The  afternoon  tub 
could  be  filled  at  5  p.  m.  and  would  be  ready  for  use  from  1  to  5  p.  m. 
of  the  following  day.  This  would  give  twenty  hours  for  the  yeast  to 
develop  to  maximum  efficiency  and  four  hours  in  which  to  use  the 
yeast. 

(i)  Simple  Yeast  Apparatus  for  Wineries. — The  simple  apparatus 
used  for  our  winery  tests  is  suitable  for  any  ordinary  cellar  and  with 
modifications  for  large  cellars. 

For  the  preliminary  operations  of  the  rejuvenation  and  increase  of 
the  pure  yeast  the  following  articles  are  needed : 

1.  A  one  quart  solid  culture  of  pure  yeast. 

2.  A  one  quart  Mason  jar  with  grape  must. 

3.  A  covered  saucepan  to  hold  the  Mason  jar. 

4.  A    small    alcohol    lamp. 

5.  A  pint  of  alhohol. 

6.  A  glass  funnel  to  fit  into  the  neck  of  a  quart  bottle. 

7.  A  1  pound  roll  of  surgeon's  cotton. 

8.  Two  quart  bottles  for  hot  water. 

9.  A  box  16  in.  by  16  in.  and  24  in.  in  depth  for  use  as  an  incubator. 

10.  Wood  wool   (excelsior)  to  fill  the  incubator. 

11.  A  blanket  to  cover  the  incubator. 

12.  A  2  gallon  demijohn  without  a  cover. 

13.  A  wash  boiler  to  hold  the  2  gallon  demijohn. 

14.  Four  small  tubs  or  vats  to  hold  50  gallons  each. 

15.  A  small  scale  to  weigh  5  pounds,  sensitive  to  %  of  an  ounce. 

16.  A  one  pint  conical  graduate. 

17.  5  pounds  of  potassium  metabisulfite. 

18.  4  two  gallon  demijohns  to  hold  the  sulfite  solution  made  by  dissolving 

10  ounces  of  potassium  metabisulfite  in  one  gallon  of  pure  water. 
One  pint  of  this  solution  will  contain  1%  ounces  of  sulfite,  which  is 
exactly  what  is  needed  to  defecate  25  gallons  of  must. 

5—230 


82 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


The  incubator,  Fig.  7,  is  a  simple  wooden  box  large  enough  to  hold  a 
two  gallon  demijohn  with  space  for  one  or  two  inches  of  excelsior 
below,  around  and  above  the  glass.     If  the  must  in  the  demijohn  is 


Fig.   6. — Apparatus  for  the  rejuvenation  of  pure  yeast. 

warm  when  put  in  the  box  and  the  whole  covered  with  a  thick  blanket, 
it  will  keep  its  heat  perfectly  in  the  coldest  night.  A  quart  bottle 
requires  the  aid  of  a  couple  of  bottles  of  hot  water.  The  water  in  these 
bottles  must  not  be  too  hot  nor  the 
insulation  too  perfect  or  the  heat 
evoked  by  the  fermenting  must  may 
raise  the  temperature  too  high  and 
injure  the  yeast. 

An  ordinary  wash  boiler  made  for 
heating  on  a  stove  is  too  small  to  hold 
a  two  gallon  demijohn  upright  but  will 
do  so  if  the  demijohn  is  inclined.  It 
is  necessary  to  make  some  kind  of 
frame  to  fit  into  the  boiler  and  hold 
the  demijohn.  This  frame  can  be  made 
of  wood  or  of  wire.  It  should  keep  the 
demijohn  from  touching  the  bottom  of 
the  boiler  to  prevent  the  direct  heat 
of  the  fire  from  cracking  the  glass.  If  the  inclination  of  the  demijohn 
is  just  sufficient  to  allow  the  cover  of  the  boiler  to  be  put  in  place,  it 
will  hold  the  needed  \\  gallons  of  must. 


Fig.   7. — Incubator  for  pure  yeast. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


83 


Two  of  the  fifty  gallon  vats  are  for  the  propagation  of  the  yeast 
starter  and  two  for  the  defecation  of  the  necessary  must.  They  should 
be  furnished  with  faucets  at  the  bottom  for  drawing  off  the  must  and 
yeast  and  with  wooden  or  canvas  covers  to  keep  out  dust.  The  defecat- 
ing vats  should  be  placed  in  such  a  position  that  the  must  can  be  run 
directly  from  them  into  the  yeast  vats.  The  arrangement  adopted  in 
our  winery  tests  is  shown  in  Fig.  8  and  was  found  satisfactory. 


Fig.  8. — Yeast  propagating  apparatus  for  a  winery. 

The  battery  of  vats  should  be  so  located  that  it  is  easy  to  supply 
them  daily  with  must  and  to  convey  the  yeast  to  the  fermenting  vats. 
It  is  convenient  to  label  these  defecating  vats  Dx  and  D2  and  the 
corresponding  yeast  vats  Yx  and  Y2. 

The  first  vat  of  starter  Y1  is  prepared  as  described  on  pages  76  to  78. 
About  12  to  24  hours  before  the  yeast  in  this  vat  is  used,  30  gallons 


84 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION, 


of  must  should  be  placed  in  vat  T>x  and  sulfited  with  1  pint  of  the 
sulfite  solution.  This  must  will  be  ready  for  vat  Yt  as  soon  as  the 
yeast  in  this  vat  has  been  used.  Before  taking  yeast  out  of  the  vat  it 
should  be  well  mixed  in  order  to  stir  up  the  yeast  which  has  settled. 
After  25  gallons  of  yeast  have  been  used,  5  gallons  remain  for  the 
inoculation  of  the  new  supply  of  must.  Twenty-five  gallons  of  must  are 
then  run  directly  from  vat  Dx  into  vat  Yx.  It  is  usually  not  necessary 
to  warm  the  must,  as  the  5  gallons  of  vigorous  young  yeast  left  in  the 
vat  insure  a  prompt  start  of  fermentation  which  will  maintain  a  favor- 
able temperature  if  the  vat  is  kept  covered.  Aeration  twice  or  three 
times  during  the  day  is  advisable  as  it  tends  to  increase  the  number 
and  vigor  of  the  yeast  cells.  Vats  Y2  and  D2  are  treated  in  the  same 
way.  By  properly  timing  the  sulfiting  in  vats  Dt  and  D2  and  the 
transference  of  the  defecated  must  from  these  vats  to  vats  Yt  and  Y„ 
a  continuous  supply  of  yeast  can  be  obtained  from  6  a.  m.  until  6  p.  m. 
The  following  table  will  show  how  this  can  be  done: 

TABLE  No.  34. 
Times  for  filling  and  using  defecating  and  yeast  vats. 


Vats. 

Time. 

*i. 

Dv 

Y2. 

D2. 

September  1,  12  m. __ 

fill 

September  1,    6  p.  m. 

fill 

September  2,  12  m.__  _ 

fill 

fill 

September  2,  6  p.  m.__ 

fill 

fill 

September  3,    6  a.  m.  _  __  _ __ 

use 
fill 

September  3,  12  m. 

fill 

use 
fill 

September  3,    6  p.  m.  _ 

fill 

September  4,    6  a.  m.  _ 

use 
fill 

September  4,  12  m 

fill 

use 
fill 

September  4,    6  p.  m 

fill 

Thus  defecating  vat  D1  is  filled  and  sulfited  at  12  m.  every  day. 
Twenty-four  hours  later  at  12  m.  of  the  following  day  the  defecated 
must  is  transferred  from  this  vat  to  yeast  vat  Yx  and  replaced  with 
fresh  sulfited  must.  Eighteen  hours  later  at  6  a.  m.  of  the  third  day 
the  yeast  in  vat  Yx  is  ready  for  use  and  can  be  employed  until  12  m. 
when  the  vat  must  be  filled  again  from  vat  Dx.  The  other  pair  of  vats  is 
treated  in  the  same  way  except  that  they  are  filled  at  6  p.  m.  and  used 
from  12  m.  to  6  p.  m. 

By  this  arrangement,  the  sulfited  must  always  defecates  for  twenty- 
four  hours  and  the  yeast  propagates  for  from  eighteen  to  twenty-four 
hours,  times  which  have  been  found  suitable  and  sufficient.  Any 
modifications  of  these  times  may  be  made  which  allow  at  least  twelve 
hours  for  defecation  and  sixteen  hours  for  fermentation.     The  defeca- 


Bulletin  230]  ENOLOGICAL   INVESTIGATIONS.  85 

tion  should  not  last  more  than  forty-eight  hours,  and  the  yeast  should 
be  used  before  the  must  becomes  quite  dry. 

A  battery  of  the  size  described  is  sufficient  to  supply  yeast  for  a 
winery  fermenting  5000  gallons  of  wine  per  day.  For  smaller  wineries 
two  vats  only  might  be  used;  for  larger  wineries  the  vats  should  be 
larger.  A  battery  consisting  of  four  500  gallon  vats  would  supply 
yeast  for  the  fermentation  of  50,000  gallons  a  day.  Such  a  battery 
should  be  supplied  with  cooling  coils,  aerating  device  and  with  hose 
and  small  electric  or  other  motor  pump  to  fill  the  defecating  vats  and 
to  transport  the  yeast  to  the  fermentation  tanks.  One  man  could  do 
all  the  work  necessary  in  running  the  largest  apparatus. 

(j)  Precautions  Necessary  for  the  Proper  Working  of  the  Yeast  Propa- 
gation. 

1.  Cleanliness.  Everything  that  comes  in  contact  with  the  yeast 
should  be  kept  clean.  Buckets,  tubs,  hose  and  pumps  used  in  handling 
the  must  and  yeast  should  be  washed  every  time  they  are  used  and 
immediately  after  using.  Rinsing  with  a  sulfite  solution  is  useful  but 
not  necessary,  if  the  utensils  are  placed  where  they  will  dry  quickly 
after  washing.  The  yeast  vats  should  be  kept  covered  both  to  keep  out 
dust  and  to  retain  the  heat. 

2.  Actions  of  the  Sulfite.  The  must  should  not  be  transferred  to 
the  yeast  vat  until  about  twelve  hours  after  sulfiting.  This  is  to  allow 
time  for  the  sulfurous  acid  to  enter  into  the  combined  form.  The  wild 
yeasts  and  other  injurious  micro-organisms  are  thus  subjected  to  the 
action  of  the  full  amount  of  free  S02  when  it  is  added,  while  the  cul- 
ture wine  yeast  comes  in  contact  only  with  a  small  amount  of  free  S02 
after  the  main  part  has  entered  into  combination.  The  consequence 
is  that  the  culture  yeast  retains  its  vigor  and  the  wild  organisms  are 
paralyzed. 

3.  Maintenance  of  Vigor  of  Yeast.  Fresh  must  should  be  added 
to  the  yeast  vats  sufficiently  often  to  keep  the  fermentation  going  con- 
tinuously. If  the  must  in  the  yeast  vats  is  allowed  to  become  perfectly 
dry  the  yeast  loses  in  vigor.  When  this  occurs  the  yeast  can  be  made 
vigorous  again  in  two  or  three  days  by.  adding  fresh  must  and  thor- 
oughly aerating. 

4.  Hastening  Development.  If  the  yeast  develops  too  slowly, 
aerate  more  and  if  necessary  warm. 

5.  Retarding  Development.  By  leaving  a  smaller  amount  of  yeast 
in  the  vat  when  adding  fresh  must,  its  development  can  be  retarded. 
This  may  be  done  when  a  day  or  more  passes  without  crushing  as  on 


86  UNIVERSITY  OP   CALIFORNIA EXPERIMENT  STATION. 

Sundays.  The  yeast  left,  however,  should  be  at  least  5  per  cent  of  the 
volume  of  the  must  added.  Less  aeration  will  also  tend  to  retard  the 
growth  of  yeast. 

6.  Plan  all  operations  so  that  the  yeast  can  be  used  at  its  maximum 
efficiency,  that  is  when  the  Balling  per  cent  has  fallen  to  between  12 
per  cent  and  2  per  cent. 


IV.    TESTS  OF  THE  USE  OF  S02  AND  PURE  YEAST  IN  A 

WINERY. 

(a)  Objects  and  Nature  of  the  Tests.  Laboratory  investigations  and 
tests  conducted  in  wineries  with  the  cooperation  of  the  owners  have 
demonstrated  thoroughly  the  utility  of  the  use  of  pure  yeast  in  com- 
bination with  sulfurous  acid  and  cooling  machines.  Many  wine- 
makers,  however,  have  an  exaggerated  idea  of  the  cost  and  trouble 
involved  in  the  use  of  devices  for  the  control  of  temperature.  The 
winery  tests  made  this  year  had  for  their  principal  objects  the  deter- 
mination of  the  possibility  of  introducing  some  of  the  benefits  of  pure 
yeasts  with  the  help  of  sulfurous  acid,  and  without  any  change  in  the 
usual  methods  of  the  winery  involving  the  installation  of  cooling 
machinery  or  other  new  appliances  except  a  simple  yeast  propagator. 

Concurrently,  observations  were  made  to  test  on  a  larger  scale  cer- 
tain conclusions  reached  by  means  of  laboratory  experiments. 

The  tests  consisted  of  parallel  series  of  fermentations,  in  one  of 
which  the  ordinary  methods  of  the  winery  were  employed,  and  in  the 
other,  sulfurous  acid  and  pure  yeast  separately  or  in  combination  were 
applied  with  as  little  change  in  the  usual  methods  as  possible.  Obser- 
vations were  made  on  the  progress  of  fermentation  and  on  the  develop- 
ment of  the  wine  so  long  as  it  was  possible  to  keep  the  various  lots 
separate.  The  winery  was  peculiarly  well  adapted  for  the  tests  pro- 
posed. It  is  situated  in  a  cool  region  where  the  need  of  temperature 
regulation  is  less  pressing  than  in  most  parts  of  California ;  the  winery 
is  well  supplied  with  the  ordinary  appliances  and  labor  saving  devices ; 
and  finally,  it  received  grapes  from  both  interior  and  coast  regions, 
which  enabled  us  to  make  our  demonstrations  on  raw  materials  of  dif- 
fering character. 

(b)  Changes  in  the  kind  and  number  of  active  micro-organisms. 
Two  vats,  each  holding  5  tons  of  grapes,  were  filled  in  the  ordinary 
way  with  Zinfandel  from  Acampo.  Experiment  171,  vat  20,  received 
the  ordinary  treatment  of  the  cellar.  Experiment  169,  vat  4,  was  sul- 
fited  by  the  addition  of  three  quarters  of  a  pound  of  potassium  metabi- 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


87 


sulfite  to  the  ton  added  gradually  as  the  vat  was  filled.  Five  hours 
after  the  addition  of  the  last  sulfite,  50  gallons  of  pure  yeast  were 
stirred  in. 

At  the  start  of  perceptible  fermentation  the  must  in  each  vat  was 
examined  by  means  of  plate  cultures.  In  the  untreated  vat  nothing 
was  found  but  molds  and  wild  yeasts;  in  the  treated  vat  nothing  but 
wine  yeast. 

Similar  tests  were  made  with  two  other  vats  with  the  following 
results : 

TABLE  No.   35. 

Effect  of  S02  and  pure  yeast  starter  on  micro-organisms. 

Experiment   198.     Vat   11.     Zinfandel  from  Contra  Costa   County. 


Before  sulflting  with 
12  ounces  per  ton. 


After  adding 

yeast 

1.5  per  cent 

starter. 


Molds 

Wild  yeasts 
Wine  yeasts 


59,400  per  c.c. 
27,700 
0 


0 

0 

all 


Experiment  202.     Vat  19.     Second  crop  Zinfandel  from  Acampo    (very  moldy). 


Sulfite  8  ounces  per  ton. 


Before  sulflting. 


After 
sulflting. 


After  adding 

1.5  per  cent 

yeast. 


Molds   — 

Apiculate  yeast 
Other  wild  yeast 
Wine  yeast  _ 


1,600,000  per  c.c. 
2,830,000 

a  few 

a  few 


0 

0 

0 

560,000 


0 

0 

0 

2,816,000 


Experiment  198  shows  that  in  must  from  normal,  sound,  clean 
grapes,  wine  yeast  exists  in  very  minute  quantities.  The  wild  yeasts, 
(principally  apiculatus)  on  the  contrary,  are  numerous.  Such  a  must 
allowed  to  ferment  spontaneously  without  the  addition  of  sulfurous 
acid  or  pure  yeast  would  not  develop  wine  yeast  in  appreciable  quan- 
tities, until  the  apiculatus  had  multiplied  sufficiently  to  seriously  affect 
the  quality  of  the  wine.  This  is  shown  by  experiment  202  where,  owing 
to  injury  to  the  grapes  and  delay  in  crushing,  the  apiculatus  yeast  has 
increased  a  hundredfold.  The  first  part  of  the  fermentation  of  all  our 
grapes  is  undoubtedly  due  to  apiculatus  when  measures  are  not  taken 
to  prevent  it.  Much  of  the  persistent  cloudiness  and  delay  in  the  finish 
of  fermentation,  even  of  otherwise  good  wines,  is  due  to  this  cause. 

The  trouble  with  apiculatus  fermentation  is  likely  to  be  worse  in  the 
cleanest  and  best  kept  wineries  and  at  the  beginning  of  the  season. 
When  crushers,  vats  and  other  surfaces,  with  which  the  grapes  come  in 
contact  are  kept  constantly  covered  with  grape  must  the  wine  yeast 


88  UNIVERSITY  OF   CALIFORNIA — EXPERIMENT   STATION. 

finally  develops  and  inoculates  the  must.  This  is  a  defective  method 
of  obtaining  wine  yeast,  however,  as  it  gives  no  assurance  of  the  kind 
of  yeast  and  introduces  also  injurious  bacteria. 

The  mold  spores,  while  even  in  good  grapes  as  numerous  as  the  wild 
yeasts,  are  innocuous  if  the  grapes  are  crushed  while  still  in  good  condi- 
tion as  they  do  not  increase  appreciably  in  the  must  or  crushed  grapes. 
If  delay  occurs  in  the  crushing,  however,  they  may  increase  rapidly 
as  shown  by  experiment  202,  in  which  case  they  may  seriously  injure 
the  wine. 

In  all  three  cases  the  injurious  molds  and  yeasts  were  eliminated 
or  paralyzed  completely,  even  in  vat  19  containing  grapes  in  very  bad 
condition.  The  number  of  wine  yeast  cells  was  so  much  smaller  than 
that  of  the  wild  forms  that  before  sulfiting  it  was  difficult  to  find  any 
at  all.  After  sulfiting,  however,  in  vat  19  they  were  found  to  exist  in 
fair  numbers.  Sulfiting  alone,  therefore,  would  have  insured  a  fer- 
mentation due  principally  to  wine  yeast  in  this  case,  though  the  molds 
and  apiculatus  yeasts  had  undoubtedly  injured  the  must  before  the 
sulfite  was  added. 

In  vat  11  containing  grapes  in  good  condition,  a  starter  of  pure 
yeast  insured  a  fermentation  due  practically  entirely  to  the  yeast 
added.  In  vat  19  the  yeast  to  which  the  fermentation  was  due  con- 
sisted of  about  four  parts  of  that  added  to  one  part  of  the  wine  yeast 
occurring  naturally  on  the  grapes.  This  shows  that  even  with  grapes 
in  very  bad  condition  much  improvement  may  be  expected  from  sul- 
fiting and  pure  yeast.  The  injurious  yeasts  are  all  eliminated  and  the 
slight  admixture  of  the  natural  wine  yeasts  can  do  no  harm. 

These  experiments  show  that  the  injurious  activities  of  molds  and 
wild  yeasts  can  be  prevented  by  prompt  crushing  and  sulfiting  followed 
by  the  addition  of  a  starter  of  good  wine  yeast. 

(c)  Duration  and  Course  of  the  Fermentation.  1.  fermentation 
curves.  "When  a  cask  of  grape  must  is  allowed  to  ferment  sponta- 
neously the  fermentation  is  not  uniform  throughout  its  course.  There 
is  first  of  all  a  period  during  which  no  fermentation  is  perceptible. 
This  is  because  the  yeasts  present  are  insufficient  to  cause  any  noticeable 
Ganges  in  the  mass.  During  this  period  the  yeasts  and  other  organ- 
isms increase  in  number  until  finally  the  mass  exhibits  signs  of  fer- 
mentation. These  signs  are  rise  of  temperature,  evolution  of  gas  and 
decrease  of  sugar  as  shown  by  the  Balling  saccharometer.  The  yeasts 
continue  to  increase  and  the  fermentation,  at  first  slow,  becomes  more 
and  more  rapid  until  it  reaches  a  maximum.  After  this  the  fermen- 
tation gradually  becomes  slower  until  it  ceases  completely.  If  it 
ceases  before  all  the  sugar  has  been  destroyed  it  is  said  to  "stick." 

The  character  of  the  fermentation  can  be  shown  graphically  by  a 


Bulletin  230]  ENOLOGICAL   INVESTIGATIONS.  89 

line  or  curve  indicating  the  rate  of  fermentation  at  all  points  of  its 
course.  Curves  of  this  kind  can  be  made  from  observations  of  the 
volume  of  gas  given  off,  of  changes  in  the  weight  of  the  fermenting 
mass,  of  the  rise  of  temperature  or  of  the  decrease  of  Balling  per  cent. 
The  changes  in  the  sugar  contents  as  shown  by  the  Balling  saccharom- 
eter  afford  a  very  simple  and  useful  means  of  forming  a  fermen- 
tation curve. 

The  shape  of  this  curve  shows  the  progress  of  the  fermentation  and 
the  changes  in  its  rapidity.  The  rapidity  of  the  fermentation  is 
influenced  by  a  number  of  factors.  It  is  the  greater  the  larger  the 
number  of  active  yeast  cells  present  and  within  certain  limits  the 
more  fermentable  matters  contained  in  the  must.  It  tends  to  decrease 
as  the  products  of  fermentation,  alcohol,  volatile  acids,  etc.,  accumulate 
in  the  must.  Aeration  tends  to  hasten  the  fermentation  by  promoting 
the  multiplication  of  the  yeast.  High  temperatures,  within  certain 
limits,  accelerate  the  rate  of  fermentation  by  increasing  the  number  of 
yeast  cells  and  rendering  them  more  active. 

A  typical  fermentation  curve  is  shown  in  Diagram  8.  This  repre- 
sents a  fermentation  of  sterilized  must  started  with  1  per  cent  of 
vigorous  yeast  and  kept  at  a  nearly  constant  temperature. 

Three  periods  may  be  distinguished,  a  fore  period  of  two  days  in 
which  the  fermentation  is  slow,  removing  an  average  of  2°  Balling 
per  day;  a  central  period  of  four  days  in  which  the  fermentation  is 
nearly  twice  as  rapid,  removing  an  average  of  3j°  Balling  per  day; 
and  an  after  period  of  3^  days  in  which  fermentation  becomes  slow 
again,  removing  an  average  of  only  LJ°  Balling  per  day.  If  the  tem- 
perature had  been  lower  or  the  amount  of  yeast  added  less,  the  whole 
fermentation  and  especially  the  fore  period  would  have  been  longer. 
If  more  aeration  had  been  given  the  whole  fermentation  and  especially 
the  central  period  would  have  been  shorter. 

Curves  of  the  fermentations  that  occur  in  practice  in  the  winery 
show  many  interesting  and  instructive  variations  of  this  type. 

The  part  showing  the  fore  period  is  usually  much  longer  and  flatter. 
This  is  because  the  amount  of  yeast  present  in  the  freshly  crushed 
grapes  is  small  and  the  temperature  low.  It  may  be  curtailed  by  the 
addition  of  a  starter  or  by  warming  the  must.  It  may  be  prolonged  by 
the  use  of  sulfites.  Occasionally  there  is  an  actual  rise  of  the  curve 
during  the  fore  period.  This  is  due  to  the  sugar  in  dried  grapes,  which 
diffuses  into  the  must  before  noticeable  fermentation  commences. 

The  part  showing  the  central  period  is  usually  shorter  and  steeper, 
particularly  in  red  wine  fermentations.  This  is  because  of  greater 
aeration  and  a  rise  in  temperature.  It  may  be  prolonged  slightly  by 
the  use  of  cooling  devices. 


90 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION. 


The  part  showing  the  after  period  often  exhibits  great  variations.  In 
very  sweet  musts  it  may  be  very  much  prolonged,  may  even  last  for 
months,  and  in  some  cases  may  become  horizontal,  showing  a  cessation  of 
fermentation  before  0  Balling0  is  reached.  This  is  due  to  the  accumula- 
tion of  products  of  fermentation  such  as  alcohol  and  volatile  acids,  which 
interfere  with  the  action  of  the  yeast.  Aeration  and  maintaining  the 
temperature  are  the  principal  means  of  preventing  the  undue  lengthen- 
ing of  this  period.  Where  the  fermentation  is  pure  and  the  tempera- 
ture is  high,  especially  in  weak  musts,  this  period  may  be  much  curtailed. 


z% 

rot 

Tr^ 

£0 

i^y/c 

)/  FN 

T  R 

^F?l( 

)D 

:   RF- 

~rrf 

?  Pr 

nia 

n 

/fi 

FTi 

Tior, 

^^ 

16 

14 

(r» 

fc 

10 

ft 

^4 

6 

<0 

4 

Z 

HC 

>URl 

zo    «?o    GO   do   /oo    /&o   /jo  teo  /SO  Z0O2Z0Z40 


Diagram  8. 
Typical  fermentation  curve  at  constant  temperature. 


Bulletin  230] 


ENOLOGICAL  INVESTIGATIONS. 


91 


2.  red  wine  fermentations.     Observations  were  made  on  the  follow- 
ing red  wine  fermentations  at  Martinez : 


Variety  and  origin  of  grapes. 

Sulfite 
per  ton. 

Pure 
yeast. 

Fermenting 
vats. 

Storage 
casks. 

I. 

Zinfandel  from  Acampo 

0 

0 

2 

! 

J 

II. 

Zinfandel  from  Acampo 

0 

0 

5 

10 

III. 

Zinfandel  from  Acampo 

5 

oz. 

plus 

3 

l 

IV. 

Zinfandel  from  Acampo 

12 

oz. 

plus 

1 

! 

j 

V. 

Zinfandel  from  Acampo 

12 

oz. 

plus 

4 

9 

VI. 

Petite  Sirah  from  Acampo 

0 

0 

9 

2 

VII. 

Petite  Sirah  from  Acampo 

8 

oz. 

plus 

7 

1 
J 

16 

VIII. 

Petite  Sirah  from  Acampo 

12 

oz. 

plus 

10 

IX. 

Zinfandel  from  Contra  Costa  Co. 

0 

0 

20 

*10-11 

X. 

Zinfandel  from  Contra  Costa  Co. 

12 

oz. 

plus 

8 

1 

12 

XI. 

Zinfandel  from  Contra  Costa  Co. 

12 

oz. 

plus 

11 

1 

XII. 

Alicante  Bouschet  from  Acampo... 

8 

oz. 

plus 

12 

j 

17 

XIII. 

Alicante  Bouschet  from  Acampo.. 

8 

oz. 

plus 

15 

J 

XIV. 

Petite  Sirah  and  Green  Hungarian 

9 

oz. 

0 

6 

18 

XV. 

Barbera  from  Contra  Costa  Co... 

5 

oz. 

0 

14 

4 

XVI. 

Zinfandel,  2d  crop  from  Acampo.. 

8 

oz. 

plus 

19 

2 

XVII. 

Alicante  Bouschet  from  Acampo.. 

8 

oz. 

0 

13 

] 

XVIII. 

Zinfandel  and  Alicante  Bouschet 
from  Contra  Costa  Co 

6.1 

8 

>  oz. 
oz. 

plus 

plus 
plus 

16 
17 

i 
l 

XIX. 

Alicante  Bouschet   from   Contra 
Costa  Co. 

26 

XX. 

Zinfandel  from  Contra  Costa  Co.. 

8 

oz. 

18 

i 
1 
J 

♦Puncheons. 


The  fermentations  were  all  conducted  in  vats  with  5-foot  staves,  hold- 
ing about  2,200  gallons.  No  cooling  was  used,  and,  as  a  consequence, 
the  temperatures  in  all  cases  were  high.  The  temperature  conditions 
of  the  locality  and  of  the  season  were  exceptionally  favorable.  The 
cellar  varied  on  the  average  from  about  61°  F.  at  night  to  about  74° 
F.,  in  the  hottest  part  of  the  day.  On  the  hottest  day  it  rose  to  84° 
F.,  and  on  the  coldest  night  it  fell  to  54°  F.  Under  less  favorable  con- 
ditions there  would  undoubtedly  have  been  more  trouble  with  ' '  stuck ' ' 
wines.  The  variations  in  the  temperature  of  the  air  of  the  fermenta- 
tion room  during  the  vintage  are  shown  %by  the  curves  of  diagram  9. 


92 


UNIVERSITY   OP    CALIFORNIA EXPERIMENT   STATION. 


8J 

/" 

T  iA/ffflMF^r  Dj9V 

80 
78 

3 

7% 
70 

kj 

IT  \nv%Finte: 

/» 

M    coolest 

1 

£ 

8s 

I 

/it 

1\ 

S^  / 

64 

l^vJSw 

m 

N^V^ 

5fi 

.■% 

f 

• 

TIMi 

c- 

i 

t>BM  IOm  MM-  &PM  4PM-  6PM  8PM  /OPMIZPMZRM  4flM  6AM MM 

Diagram  9. 
Variation  in  temperature  of  fermenting  room. 

The  grapes,  after  stemming  and  crushing,  were  conveyed  by  means 
of  a  must  pump  directly  to  the  fermenting  vats.  The  sulfurous  acid 
was  added  in  fractional  doses  gradually  as  the  vats  were  filled  and 
mixed  by  repeated  stirring.  The  sulfurous  acid  was  used  in  the  form 
of  a  strong  water  solution  of  potassium  metabisulfite.  After  allowing 
the  sulfited  grapes  to  stand  for  some  hours,  30  to  40  gallons  of  yeast 
were  added.  This  represents  a  starter  of  from  1J  to  2  per  cent.  The 
yeast  was  mixed  as  thoroughly  as  possible  by  means  of  punching. 
A  better  method  would  have  been  to  pump  over  the  must,  as  soon  as 
the  vat  was  filled,  to  distribute  the  sulfite  equally  and  again  while  add- 
ing the  yeast. 

The  vats  were  kept  covered  until  the  temperature  began  to  rise,  when 
the  covers  were  removed  and  punching  practised  three  times  a  day. 
When  the  temperature  commenced  to  fall,  the  covers  were  replaced 
unless  the  vats  were  drawn  off  immediately.  In  most  cases  the  draw- 
ing off  was  delayed  until  the  Balling  had  fallen  to  0°.  A  record  was 
kept  of  the  disappearance  of  the  sugar  and  of  the  changes  in  tempera- 
ture. 

The  first  series  of  fermentations  was  made  with  Zinfandel  grapes 
from  Acampo,  San  Joaquin  County.  The  grapes  were  in  fair  to  poor 
condition,  the  different  lots  containing  a  smaller  or  larger  proportion 
of  dried  and  moldy  grapes.  Two  vats  were  allowed  to  ferment  in  the 
ordinary  way  without  sulfite  or  starter.     One  vat  was  sulfited  at  the 


Bulletin  230] 


EXOLOGICAL   INVESTIGATIONS. 


rate  of  5  ounces  to  the  ton,  and  two  at  the  rate  of  12  ounces, 
sulfited  vats  were  started  with  pure  yeast. 


93 
All  three 


TABLE  No.   36. 

Fermentation   I.     Zinfandel  from   Acampo,  Vat  No.   2,  ordinary   fermentation,  no 

sulfite,  no  starter. 


Balling 

per  cent. 

Temperature. 

Date. 

Hour. 

Cap- 

Bottom.  |      Room. 

Remarks. 

Oct.   24 

Oct.   24 

Oct.  25 
Oct.   25 

Oct.  26 

Oct.  26 
Oct.  27 
Oct.  27 

8       a.m. 
2       p.m. 

7  a.  m. 

8  p.  m. 

8  a.m. 

9  p.m. 

8  a.m. 

9  a.m. 

2:30  p.m. 
8       p.m. 
8       a.  m. 

12           m. 

8       p.m. 

8  a.m. 
11       a.m. 

4  p.m. 
10:30  p.m. 

23.6 

23.7 

24.1 
24.6 

24.8 

24.2 
24.1 



59.0 

59.0 

57.0 
60.0 

57.0 

59.0 
59.0 

54.5 

69.0 

54.5 
64.5 

61.0 

Started  to  fill  vat.     Grapes 

show  many  raisins. 
Vat  half  full.    Grapes  a  little 

cleaner  than  in  Vat  1. 
Slight  signs  of  fermentation 
Slow  fermentation.    Smell  of 

CO2  gas. 
Slow  fermentation.    Smell  of 

CO2  gas. 
Gas  coming  off  freely. 
Fermentation  vigorous. 
Started   to    complete   filling 

Oct.   27 

of  vat. 
Vat  full. 

Oct.  27 
Oct.  28 

Oct.  28 

Oct.   28 

Oct.   29 
Oct.  29 
Oct.  29 
Oct,   29 

22.7  • 
22.1 

20.5 

16.0 

5.5 
2.7 
1.6 

0 

64.5 
70 

70 

80 

97 
99.5 
100.5 

73.5 
66.0 

69.0 

78.0 

91.5 

"l00~5~ 
97.0 

61.0 
59.0 

71.5 

70.5 

59.0 
71.5 
72.5 

Fermentation  of  the  grapes 
first  crushed  has  raised  the 
temperature  of  the  bottom 

Temperature  taken  after 
punching. 

Temperature  taken  after 
punching. 

Fermentation  subsiding. 
Drew  off  free  run  into  cask  10. 

The  rise  of  Balling  per  cent  from  23.6  to  24.8  during  the  first  forty- 
eight  hours  is  due  to  the  presence  of  numerous  dried  grapes.  It  is  not 
considered  good  practice  to  spread  the  filling  of  a  vat  over  two  or  three 
days,  as  in  this  case,  although  it  is  said  to  promote  the  dissipation 
of  some  of  the  heat  of  fermentation  where  cooling  devices  are  not 
employed.  This  tendency  is  very  slight,  however,  as  the  tempera- 
ture in  fermentation  II  only  rose  about  two  degrees  higher  than  in  I, 
although  the  original  temperature  of  the  grapes  was  about  12  degrees 
higher. 


94 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 


TABLE  No.   37. 

Fermentation  II.     Zinfandel  from  Acampo,  Vat  No.  5,  ordinary  fermentation, 
no  sulfite,  no  pure  yeast. 


Hour. 

Balling 
per  cent. 

1  emperature. 

Date. 

Cap. 

Bottom. 

Room. 

Remarks. 

Oct.  29 

10  a.  m. 
6  p.  m. 

Began  filling  vat. 

Oct.  29 

Vat  filled  and  covered. 

Oct.  29 
Oct.  30 
Oct.  30 
Nov.    1 
Nov.    1 
Nov.    1 

8  p.m. 
8  a.  m. 
8  p.m. 
8  a.m. 
12      m. 

8  p.m. 

9  a.m. 
12      m. 
10  p.  m. 

8  a.m. 
8  p.m. 
8  a.m. 

21.7 

22.0 

22.5 

23.0 

22.5 

22.0 

17.0 

12.0 

4.0 

1.0 

.3 

.2 

""68.6" 

68.0 

71.5 
64.5 
64.5 
69.0 
69.0 

70.5 
61.5 
71.5 
62.5 
71.5 
64.5 
59.0 
68.0 
68.0 
59.0 
57.0 
61.0 

Slight  signs  of  fermentation. 

Slow  fermentation. 
Slow  fermentation. 
Fermentation  well  started. 

Nov.    2 
Nov.    2 
Nov.    2 
Nov.    3 
Nov.   3 
Nov.    4 

79.0 

84.0 

100.5 

102.0 

100.5 

97.0 

77.0 
82.5 
99.5 
101.5 
100.5 
97.0 

Fermentation  vigorous. 

Fermentation  slackening. 
Drew  off  free  run  into  cask  10. 

This  vat  also  shows  a  rise  in  Balling  in  the  first  forty-eight  hours,  due 
to  the  presence  of  dried  grapes. 


TABLE  No.  38. 

Fermentation  III.     Zinfandel  from  Acampo,  Vat  No.  3,  sulfite  5  ounces  per  ton, 

pure  yeast. 


Hour. 

Balling 
per  cent. 

Temperature. 

Date. 

Cap. 

Bottom. 

Room. 

Remarks. 

Oct.  27 

2       p.  m. 

Started  filling  vat. 

Oct.  27 

3  p.m. 

4  p.  m. 
6       p.m. 
6:15  p.m. 
6:30  p.m. 

10:30  p.m. 

8       a.m. 

12          m. 

8      p.m. 

8  a.m. 
12          m. 

8       p.m. 

8  a.m. 
12          m. 

6      p.m. 

8      p.m. 

8  a.m. 
10:39  a.m. 

4      p.m. 

Added  8  ounces  sulfite. 

Oct.  27 

Added  8  ounces  sulfite. 

Oct.  27 

Added  16  ounces  sulfite. 

Oct.  27 

Added  8  ounces  sulfite. 

Oct.  27 

Added  5  ounces  sulfite. 

Oct.  27 
Oct.  28 
Oct.  28 

Oct.  28 
Oct.  29 
Oct.  29 
Oct.  29 
Oct.  30 
Oct.  30 
Oct.  30 
Oct.  30 
Nov.    1 
Nov.    1 
Nov     1 

~"~22~3~ 
22.3 

22.3 

22.2 

20.2 

16.2 

14.9 

11.5 

6.7 

5.0 

1.2 

.9 

.3 

66.0 

~~~7lT 

82.5 
82.5 
88.0 
89.5 
97.0 
100.5 

67.0 
67.0 
66.0 

66.0 
68.0 

~~~75~6~ 

86~6" 

86.0 

97.0 

100.5 

100.5 

61.0 
60.0 

71.0 
61.0 
70.0 
70.0 
61.5 
76.0 
68.0 
73.5 
62.5 
64.5 

Added  starter  of  yeast. 
No  visible  fermentation. 
Fermentation    started    very 
slightly. 

Fermentation  well  started. 
Fermentation  vigorous. 

Drew  off  free  run  into  cask  1. 

Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


95 


TABLE  No.   39. 

Fermentation    V.     Zinfandel   from   Acampo,   Vat.   No.   k;   sulfite,  12   ounces   per   ton, 

pure  yeast. 


Hour. 

Balling 
per  cent. 

Temperature. 

Date. 

Cap. 

Bottom. 

Room. 

Remarks. 

Oct.  23     10       a.m.        21.9 

Started   filling   vat;    grapes 

Oct.  23     10:30  a.m. 

I 

very  moldy. 
Added  8  ounces  sulfite. 

Oct.  23     11:30  a.m. 
Oct.  23       2:30  a.m. 

23.2 



64.5 

Added  24  ounces  sulfite. 
Added  32  ounces  sulfite. 

Oct.  23      6       p.m. 

Added  40  ounces  sulfite. 

Oct.  24       9       a.m. 
Oct.  24       9       p.m. 
Oct.  25       7:30  a.m. 
Oct.  25      8:30  a.m. 

22.5 
22.5 
22.5 

64.5 
64.5 
62.5 

54.5 
64.5 
54.5 

No  fermentation. 

A  few  bubbles  of  COa. 

Added  15  ounces  sulfite. 

Oct.   25     11 :30  a.m. 

22.5 
23.5 
23.6 
23 
22.7 
22.1 
20.5 
16.3 
11.2 
5.3 
2.5 

66.0 
64.0 
64.5 
61.0 

~"~6l"6" 

60.0 
71.5 
70.5 

Added  50  gallons  pure  yeast. 

Oct.  25 
Oct.  26 
Oct.  26 
Oct.  27 
Oct.  27 
Oct.  27 
Oct.  28 
Oct.  28 
Oct.  28 
Oct.  28 
Oct.  28 

8      p.m. 

1       p.m. 

9:45  p.m. 

7:45  a.m. 
11:40  a.m. 

8      p.m. 

8      a.m. 
12          m. 

8      p.m. 
12      p.  m. 

1       a.  m. 

"""70.5" 

""~7"o"6" 

74.5 
95.0 

"Tool" 

100.5 

68.0 
68.0 
68.0 
68.0 
68.0 
71.5 
82.5 
84.0 
97.0 
98.5 

Slight  fermentation. 
Fermenting  slowly. 
Fermenting  well. 

Removed  cover. 

Drew  off  into  cask  9. 

Oct.  28       2:30  a.m. 

2.7 
1.8 

.7 

97.0 

Oct.  29 
Oct.  29 

8       a.m. 
11:30  a.m. 

99.5 
100.5 

70.0 

This  fermentation  shows  very  clearly  the  ineffectiveness  of  S02  in 
controlling  temperature.  The  temperature  reached  97°  F.,  while  the 
must  still  showed  over  5°  Balling.  The  wine,  however,  continued  to 
ferment  and  became  dry.  Without  the  sulfite  it  would  probably  have 
" stuck"  and  remained  sweet. 


TABLE  No.  40. 

Fermentation  IV.     Zinfandel  from  Acampo,   Vat.  No.  ll;    sulfite,  12  ounces  per  ton; 

pure  yeast. 


Balling 
per  cent. 

Temperature. 

Date.                 Hour. 

Cap. 

Bottom. 

Room. 

Remarks. 

Oct.  28 

8       a.m. 
10:30  a.m. 

3      p.m. 
8      a.m. 
8      p.m. 
8       a.m. 
8      p.m. 

7  a.m. 
12          m, 
10       p.  m. 

8  a.m. 
2       p.m. 

Commenced  to  fill  vat. 

Oct.  28 

Vat  filled;  1\  pounds  of  sul- 

Oct. 29 

22.7 

22.7 

22.3 

22 

20 

16 

12 

3.5 

0 

fite  added  in  all. 
Added  40  gallons  pure  yeast. 

Oct.  30 
Oct.  30 
Nov.    1 
Nov.    1 
Nov.    2 
Nov.    2 
Nov.    2 
Nov.    3 
Nov.    3 

7L5~ 

70.5 
68.0 
89.5 
84.0 
98.5 
100.5 

66.0 
64.5 
68.0 
68.0 
71.5 
82.5 
97.0 
100.5 

61.5 
71.5 
62.5 
64.5 
61.0 

"~~71~5~ 
59.0 

Slight  signs  of  fermentation. 
Vigorous   fermentation. 

Drew  off  free  run  into  cask  9. 

96 


UNIVERSITY    OK    CALIFORNIA EXPERIMENT   STATION 


The  foregoing  tables  and  diagram  10,  made  from  them,  afford  us  a 
means  of  judging  of  the  influence  of  sulfiting  and  starters  on  the  course 
of  the  fermentations.  A  comparison  of  the  five  fermentations  is  made 
in  Table  41. 


TABLE    No.    41. 


Co 

mparison  of  fermentations  with  and  ' 
Summary  of  Fermentations  I, 

without  S02  and  pure  yeast. 
II,  III,  IV  and  V. 

Balling 
per 
cent. 

Temp. 

Sulfite. 

Yeast. 

Fermentation. 

Sugar  lost  per 
24  hours. 

Com- 
menced. 

Lasted. 

Total. 

A 

From 
filling. 

B 

From 
start- 
ing. 

c 

From 
visible 
fer- 
ment. 

I   

II   

III   

IV 

V 

24.8 
23.0 
22.3 
23.6 

22.7 

59.0 

71.6 
67.1 
64.4 
66.0 

0 

0 

5  oz. 
12  oz. 
12  oz. 

0 

0 

4  hrs. 
50  hrs. 
29  hrs. 

52  hrs. 
36  hrs. 
34  hrs. 
67  hrs. 
50  hrs. 

82  hrs. 
72  hrs. 
58  hrs. 
79  hrs. 
68  hrs. 

134  hrs. 
108  hrs. 
92  hrs. 
146  hrs. 
118  hrs. 

4.44 

5.11 

5.82 
3.88 
4.62 

4.44 
5.11 

6.08 
5.90 
6.12 

7.26 
6.67 
9.23 
7.17 

8.01 

The  third  column  from  the  end  (A)  shows  the  influence  of  the  com- 
bination of  sulfite  and  a  starter  on  the  total  time  of  fermentation.  Dur- 
ing the  134  hours,  which  the  fermentation  of  No.  I  lasted,  an  average 
of  4.44  per  cent  Balling  was  lost  every  twenty-four  hours.  No.  II 
required  less  time,  5.11  per  cent  Balling  disappearing  in  twenty-four 
hours.  This  difference  was  due  to  the  higher  temperature  of  the  grapes 
of  No.  II  when  crushed. 

In  No.  Ill,  5.82  per  cent  Balling  disappeared  in  twenty-four  hours, 
which  is  more  than  shown  by  either  untreated  vat.  This  shows  that 
the  delay  due  to  a  small  dose  of  sulfite  is  less  than  the  gain  in  time,  due 
to  the  addition  of  yeast,  if  the  starter  is  added  soon  after  the  sulfiting. 

In  No.  IV  fifty  hours  elapsed  from  the  crushing  of  the  grapes  until 
the  addition  of  the  starter.  The  use  of  the  starter  did  not  compensate 
for  this  loss  of  time  and  only  3.88  per  cent  Balling  disappeared  in 
twenty-four  hours.  In  No.  V,  twenty-nine  hours  were  allowed  to  elapse 
between  the  sulfiting  and  the  addition  of  the  starter,  and  the  sugar  dis- 
appeared at  the  rate  of  4.62  per  cent  Balling  in  twenty-four  hours. 
This  rate  is  intermediate  between  the  two  untreated  vats,  and  is  just 
about  what  might  have  been  expected  from  the  original  temperature 
of  the  grapes,  which  is  also  intermediate  between  those  of  the  untreated 
vats. 

These  results  indicate  that  a  delay  of  the  fermentation  of  twenty- 
four  hours,  due  to  sulfiting,  can  be  overcome  by  the  use  of  a  starter. 
As  other  results  show  that  the  starter  is  best  used  within  six  to  twelve 
hours  after  sulfiting,  the  combination  of  sulfite  and  pure  yeast  gives 
a  net  gain  in  the  time  of  fermentaion.     This  gain  is  due  to  the  time 


Bulletin  230]  ENOLOGICAL    INVESTIGATIONS.  97 

needed  by  the  natural  yeast  of  the  grapes  to  multiply  sufficiently  to 
cause  perceptible  fermentation. 

In  cellars  where  the  vats,  crushers  and  conveyors  are  allowed  to 
remain  covered  with  grapes  and  must  they  may  supply  a  natural 
starter.  If  this  starter  happens  to  be  principally  wine  yeast,  the  con- 
sequent fermentation  may  finish  sooner  than  that  of  a  sulfited  vat 
started  with  pure  yeast.  If  the  natural  starter,  however,  as  will  usually 
be  the  case,  contains  a  large  proportion  of  injurious  organisms  the  fer- 
mentation may  be  much  longer,  owing  to  delay  in  the  latter  part. 

The  second  column  from  the  end  (B)  shows  the  rate  of  disappear- 
ance of  the  sugar  after  the  addition  of  the  starter  in  III  and  IV  and  V. 
If  we  compare  these  figures  with  those  of  I  and  II  in  the  third  column 
from  the  end,  we  eliminate  the  effect  of  the  sulfite,  and  have  a  measure 
of  the  effect  of  the  starter  alone.  This  indicates  a  considerable  increase 
in  the  rate  of  fermentation.  Basing  the  calculation  on  a  comparison 
of  No.  IV  with  the  mean  of  Nos.  I  and  II,  we  have  an  increased  rate 
of  fermentation  of  about  23  per  cent. 

Finally,  in  the  last  column  we  have  some  light  on  the  influence  of 
the  sulfite  alone.  The  figures  give  the  average  loss  of  sugar  per  twenty- 
four  hours  from  the  start  of  visible  fermentation  to  its  completion  at 
0  per  cent  Balling.  The  most  rapid  loss  is  in  No.  Ill,  which  received 
a  small  dose  of  sulfite.  No.  II  in  spite  of  the  higher  temperature  of 
the  grapes,  fermented  at  a  rate  about  17  per  cent  slower.  This  might 
indicate  a  superiority  of  the  added  pure  yeast,  but  more  probably  indi- 
cates that  in  No.  II  the  growth  of  wild  yeasts  diminished  the  activity 
of  the  natural  wine  yeast.  Nos.  IV  and  V,  which  received  a  larger 
dose  of  sulfite,  fermented  at  about  the  same  rate  as  Nos.  I  and  II,  which 
received  none.  This  seems  to  indicate  that  the  weakening  effect  in  the 
yeasts  of  such  doses  of  S02  was  in  this  case  about  equal  to  that  of  the 
wild  yeasts,  so  far  as  the  activity  of  the  fermentation  is  concerned.  The 
effect  on  the  quality  of  the  wine,  however,  is  quite  different.  The  wild 
yeasts  depreciate  the  quality  of  the  wine,  and  may  prevent  the  com- 
pletion of  the  fermentation.  No  such  effects  have  been  noted  to  follow 
the  use  of  these  doses  of  S02. 

A  comparison  of  these  five  fermentations  is  made  by  the  curves  of 
diagram  10.  The  time  of  the  addition  of  the  pure  yeast  starter  is  shown 
at  the  right  of  the  diagram.  The  curve  beyond  this  time  should  be  com- 
pared with  the  whole  curve  of  the  unsulfited  vats.  The  whole  work  of 
the  yeast  is  shown  in  this  way  to  have  required  from  4J  to  6  days  in  the 
unsulfited  vats  and  from  3£  to  4  days  in  the  sulfited.  The  tumultuous 
fermentation,  however,  was  more  rapid  in  the  unsulfited,  as  shown  by 
the  steeper  curve  representing  this  stage. 

6—230 


3- 


i£^tPm^^m^F%  j»  ,b* 


HOUFfS 


Diagram  10. 
Fermentation  curves  of  Acampo  Zinfandel. 


Bulletin  230] 


ENOLOGICAL    INVESTIGATIONS. 


99 


TABLE  No.  42. 
Fermentation  VI.     Petite  Sirah  from  Acampo,  Vat  No.  9,  no  sulfite,  no  starter. 


Hour. 

Bailing 
per  cent. 

Temperature. 

Date. 

Cap. 

Bottom. 

Room. 

Remarks. 

Nov.    6 

9       a.m. 
6      p.m. 
8       a.m. 
1       p.m. 
6      p.m. 
8       a.m. 
1       p.m. 
6      p.m. 
8      p.m. 
1       p.m. 
6      p.m. 
8       a.m. 
1       p.m. 
5:30  p.m. 
8      a.m. 

Commenced  to  fill  vat. 

Nov.    6 
Nov.    7 
Nov.    7 
Nov.    7 
Nov.    8 
Nov.    8 
Nov.    8 
Nov.    9 
Nov.    9 
Nov.    9 
Nov.  10 
Nov.  10 
Nov.  10 
Nov.  11 

21.0 

21.2 

22.5 

22.1 

21.0 

20.0 

20.8 

18.0 

16.0 

10.0 

4.5 

1.5 

.5 

0 

"""62.fi" 

~~~68~6~ 
68.0 
70.0 
80.5 
84.0 

""Trib'i" 

100.5 
95.0 

72.5 
61.5 
62.5 
62.5 
65.5 
67.0 
68.0 
68.0 
80.5 
82.5 
91.5 
98.5 
100.5 
97.0 

72.5 
60.0 
71.5 
73.5 
58.0 
71.5 
71.5 
62.5 
66.0 
68.0 
57.0 
70.0 

Fermenting  slowly. 
Fermenting  vigorously. 

Drew  off  into  cask  2. 

TABLE  No.  43. 

Fermentation  VII.     Petite  Sirah  from  Acampo,  Vat  No.  7;    sulfite,  8  ounces  per  ton 

pure  yeast. 


Balling 
per  cent. 

Temperature. 

Date.                 Hour. 

1 
Cap.         Bottom. 

Room. 

Remarks. 

Nov.   4 

1       p.m.        21.0 

Started  to  fill  vat  and  added 

Nov.    4 

5      p.  m. 

4  pounds  sulfite. 
Finished  filling  gradually. 

Nov.    5 
Nov.    5 

9       a.m. 
1:30  p.m. 
6      p.m. 
8       a.m. 
1       p.m. 
6      p.m. 
8       a.m. 
1       p.m. 
6      p.m. 

8  a.m. 

9  a.m. 

22.5 

59.0 

61.0 
63.5 
71.0 
60.0 
70.0 
73.5 
58.0 
75.0 
71.5 
70.0 

Added  30  gallons  of  yeast. 

Nov.    5 
Nov.    6 
Nov.    6 
Nov.    6 
Nov.    7 
Nov.    7 
Nov.    7 
Nov.    8 
Nov.    8 

22.4 

"~2~f.6~ 

20.0 

13.0 

6.0 

1.75 

0 

""67"" 
86.0 

""96.6" 
100.5 
100.5 

62.5 

~~~64~5" 
73.5 
88.5 
97.0 
97.0 
98.5 

Fermentation  started. 
Fermenting  well. 
Fermentation  vigorous. 

Drew  off  free  run  into  cask  16. 

TABLE  No.   44. 

Fermentation  VIII.     Petite  Sirah  from  Acampo,  Vat  No.  10;  sulfite,  12  ounces  per  ton. 

pure  yeast. 


i 

Balling 
per  cent. 

Temperature. 

Date.                 Hour. 

Cap. 

Bottom. 

Room. 

Remarks. 

Nov.    7      1:30  p.m. 

Began  filling  vat  and  adding 

sulfite  gradually. 
Vat  full. 

Added  pure  yeast. 
Fermentation  starting. 
Fermentation  vigorous. 

Drew  off  into  cask  16. 

Nov.    7       5:20  p.m. 

Nov.    8      8       a.m. 
Nov.    8      6      p.m. 
Nov.    9      8       a.m. 
Nov.    9      1       p.m. 
Nov.    9       6       p.m. 
Nov.  10      8       a.  m. 
Nov.  10      1       p.  m. 
Nov.  10      5:30  p.m. 

21 

21.75 

21 

16 

10.5 

4 

1.5 
.25 

~~"8~9~5~ 
97.0 

102.0 
102.0 

66.6 
71.5 
68.0 
84.0 
83.5 
97.0 
98.5 
98.5 

58.0 
75.0 
62.5 
66.0 
68.0 
57.0 
70.0 
68.0 

100  UNIVERSITY  OF  CALIFORNIA — EXPERIMENT  STATION. 

TABLE  No.  45. 
Summary  of  Fermentations  VI,  VII  and  VIII. 


Balling 
per 
cent. 

Temp. 

Sulfite. 

Pure 
yeast. 

Fermentation. 

Sugar  lost  per 
24  hours. 

Com- 
menced. 

Lasted. 

Total. 

A 

From 
filling. 

B 

From 
starter. 

c 

From 
visible 
fer- 
ment. 

VI   

VII   

VIII   

21 

22.5 

21.0 

72.5 

59 

66 

0 

8oz. 
12  oz. 

0 

18  hrs. 
15  hrs. 

36  hrs. 
25  hrs. 
25  hrs. 

74  hrs. 
62  hrs. 
55  hrs. 

110  hrs. 
87  hrs. 
80  hrs. 

4.58 
6.21 

6.30 

4.58 
7.83 
7.76 

6.80 
8.71 
9.17 

The  results  shown  in  Table  45  confirm  the  conclusions  drawn  from 
those  of  Table  41.  Column  A  shows  a  net  gain  of  over  35  per  cent  in 
the  rapidity  with  which  the  sugar  disappears,  in  favor  of  the  vats 
receiving  sulfite  and  pure  yeast.  This  indicates,  as  in  the  previous 
experiments,  that  a  delay  of  eighteen  hours,  due  to  sulfiting,  is  more 
than  compensated  for  by  the  gain  in  time,  due  to  the  addition  of  a 
starter  after  that  delay. 

The  gain,  due  to  the  starter  alone,  as  indicated  by  column  B,  is  about 
70  per  cent.  Column  C  shows  that  the  pure  yeast  fermented  about  32 
per  cent  faster  in  the  sulfited  must  than  the  mixed  natural  yeasts  did 
in  the  unsulfited. 

The  character  of  the  three  fermentations  is  shown  graphically  in 
diagram  11.  Both  the  sulfited  vats  finished  their  fermentation  before 
the  witness,  although  the  grapes  were  much  colder  when  crushed. 
No.  VIII  finished  before  No.  VII,  because  the  grapes  were  warmer  and 
not  quite  so  sweet.  There  is  no  evidence  that  the  larger  amount  of 
sulfite  delayed  the  fermentation  at  all. 

All  three  fermentations  continued  until  the  Balling  saccharometer 
indicated  0.  If  the  weather  had  been  warm,  it  is  very  probable  that 
the  unsulfited  vat  would  have  failed  to  finish. 


35 ^8 BO 75T 

Diagram  11. 
Fermentation  curves  of  Petite  Sirah ;  VI,  VII,  VIII. 


Bulletin  230] 


ENOLOGICAL    INVESTIGATIONS. 


101 


TABLE  No.  46. 

Fermentation  IX.     Zinfandel  from  Contra  Costa  County,  Vat  No.  20,  ordinary 
fermentation,  no  sulfite,  no  starter. 


Temperature. 

Date. 

Hour-             per"  cent. 

Cap.         Bottom. 

Room. 

Remarks. 

Oct.  23     10       a.m.         24.5 

Good  condition;  good  many 

Oct.  24 
Oct.  25 

raisins  (first  load). 
Second  load;  reduced  sugar. 



1J   loads;   fermentation  vig- 

Oct. 25 
Oct.  26 
Oct.  26 
Oct.  26 
Oct.  27 
Oct.  27 

6      p.m. 

8  a.m. 
11       a.m. 

6      p.m. 

8  a.m. 
11:30  a.m. 

22.0 
19.0 
17.0 
14.5 
6.0 
4.5 

73.5 

75.0 

81            79.0 
86           84.0 
97           95.0 

72.0 
60.0 
64.0 
65.0 
59.0 

orous. 
Drew  off  2  puncheons;  rest  of 

Oct.  27  1    6       p.m. 

3.5 
1.0 

.25 

record    refers    to    wine    in 

Oct.  28      6      p.m. 

puncheons. 

Oct.  30      8       a.m. 

Nov.    4   i        0 



TABLE  No.  47. 

Fermentation  X.     Zinfandel  from  Contra  Costa  County,  Vat  No.  8;   sulfite  12  ounces, 

pure  yeast. 


Balling 
per  cent. 

Temperature. 

Date.                 Hour. 

Cap. 

Bottom. 

Room. 

Remarks. 

Oct.     4 

4  p.m. 
2      p.m. 

5  p.m. 

10:30  p.m. 

2      p.m. 
5:15  p.m. 

6  p.m. 
7:30  a.m. 
6      p.m. 
8       a.m. 
1       p.m. 
6      p.m. 
8      a.m. 

12          m. 
5      p.m. 

24 

First  load  crushed;  added  2 

Oct.     5 

pounds  of  sulfite. 
Second  load  crushed;  added  2 

Oct.     5 

pounds  of  sulfite. 
Third  load  crushed;  added  1 

Oct.     6 

23.8 

pound  of  sulfite. 
Fourth  load  crushed;  added 

Oct.     6 

2  pounds  of  sulfite. 
Added  2  pounds  sulfite. 

Oct.     6 

Finished  filling  and  added  2 

Oct.     6 
Oct.     7 
Oct.     7 
Oct.     8 
Oct.     8 
Oct.     8 
Oct.     9 
Oct.     9 
Oct.     9 

22.3 

22 

21 

19.3 

15 

11 

3 

2 

0 

"Too"" 

68 
70 
70 
73 

83 
86 
99 
100 
98.5 

73 
60 
70 
58 
75 
72 
70 
66 
68 

pounds  sulfite.     Reduced 
sugar. 
Added  pure  yeast. 

Fermenting. 

Very  active. 

Drew  off. 

102 


UNIVERSITY  OF  CALIFORNIA — EXPERIMENT  STATION. 


TABLE  No.  48. 
Fermentation  XI.     Zinfandel  from  Contra  Costa  County,  Vat  No.  11;  sulfite,  U  ounces; 

pure  yeast. 


Temperature. 

Date. 

Hour. 

[  per  cent. 

Cap. 

Bottom. 

Room. 

Remarks. 

Oct.     9 

10      a.  m. 

Began  crushing;  2|  pounds 
sulfite  added  gradually. 

Continued  crushing;  three 
pounds  sulfite  added  grad- 
ually. 

Continued  crushing;  two 
pounds  sulfite  added  grad- 
ually. 

Finished  crushing;  many 
raisins. 

Reduced    sugar    and    added 

Oct.  10 

Oct.  11 

Oct.  11 

3:30  a.m. 
6      p.m. 

8      a.m. 

1  p.m. 
6      p.m. 
8      a.m. 

2  p.m. 
6      p.m. 
8      a.m. 
1      p.m. 
6      p.m. 
8      a.m. 

Oct.  11 

23.5 

22 

21.8 

20.5 

18 

15 

11.5 

6 

4 

2.5 

Oct.  12 
Oct.  12 
Oct.  12 
Oct.  13 
Oct.  13 

----- 

64 
70 
69 
74 

61 

67 
74 
61 
71 
68 
63 
73 
77 
58 

pure  yeast. 
Fermentation  started. 
Fermenting  actively. 

Oct.  13 
Oct.  14 
Oct.  14 
Oct.  14 
Oct.  15 

84 
""99"" 

83 
97 
97 
99 

Drew  off. 

&3v 


X*. 


~k  ~-%£  zt§  -#F"  60     /W     S-F     9b     JO8 

Diagram  12. 
Fermentation  curves — Zinfandel  from  Contra  Costa  County. 

The  grapes  used  in  this  series  were  in  good  condition,  clean  and  none 
moldy.  They  were  thoroughly  ripe  and  included  a  few  raisins.  The 
vats  were  filled  gradually  as  the  loads  arrived,  about  fifty  hours  elaps- 
ing between  the  commencement  and  the  end  of  filling.  The  sulfited 
vats  were  prevented  from  fermenting  until  the  filling  was  complete. 
The  unsulfited  vat  commenced  to  ferment  vigorously  before  the  last 
loads  were  crushed.  The  violent  fermentation  of  the  sulfited  vats, 
therefore,  took  place  fifteen  or  twenty  hours  later  than  that  of  the  wit- 
ness vat.    In  spite  of  this  delay  the  sulfited  vats  became  dry  sooner. 


Bulletin  230] 


ENOLOGICAL    INVESTIGATIONS. 


103 


In  neither  of  the  sulfited  vats  was  there  any  perceptible  fermenta- 
tion before  the  addition  of  the  starter  of  pure  yeast.  This  shows  the 
practicability  of  delaying  fermentation  of  crushed  grapes  forty-eight 
hours  by  the  use  of  sulfite,  and  their  perfect  subsequent  fermentation 
by  means  of  pure  yeast.  Where  grapes  have  to  be  transported  long 
distances  from  the  vineyard  to  the  cellar,  it  would  undoubtedly  be  bet- 
ter to  crush  and  sulfite  them  at  the  vineyard  and  transport  them  in  this 
condition  to  the  winery.  The  activities  of  molds  and  vinegar  bacteria, 
so  injurious  to  wine  grapes  transported  in  the  usual  way  in  flat  cars 
or  boxes,  would  thus  be  prevented. 

Eecords  similar  to  the  foregoing  were  kept  on  all  the  red  wine  fer- 
mentations with  similar  results.  All  showed  the  prompt  start  and 
quicker  finish,  due  to  the  addition  of  pure  yeast.  They  showed  also 
that  sulfites  in  the  amounts  used  did  not  delay  the  fermentation  appre- 
ciably, and  were  of  no  value  in  preventing  hot  fermentations,  though 
they  may  and,  in  fact,  do  in  many  cases  prevent  some  of  the  worst  con- 
sequences of  hot  fermentations  by  discouraging  the  growth  of  bacteria. 

3.  White  Wine  Fermentations. — Observations  were  made  on  the 
following  white  wine  fermentations : 


Variety  and  origin  of  grapes. 

Sulfite 
per  ton. 

Pure  yeast. 

Fermenting  vessel. 

I. 

Green  Hungarian  from  Acampo__ 

0 

0 

puncheon  No.   1 

II. 

Green  Hungarian  from  Acampo__ 

0 

0 

puncheon  No.   8 

III. 

Green  Hungarian  from  Acampo__ 

0 

0 

puncheon  No.  9 

IV. 

Green  Hungarian  from  Acampo__ 

12  oz. 

0 

puncheon  No.   4 

V. 

Green  Hungarian  from  Acampo__ 

12  oz. 

plus 

puncheon  No.  2 

VI. 

Green  Hungarian  from  Acampo__ 

12  oz. 

plus 

puncheon  No.  3 

VII. 

Green  Hungarian  from  Acampo— 

12  oz. 

plus 

puncheon  No.  6 

VIII. 

Green  Hungarian  from  Acampo.. 

12  oz. 

plus 

puncheon  No.  7 

IX. 

Green  Hungarian  from  Acampo__ 

12  oz. 

plus 

1500-gal.  cask  No.   7 

X. 

Palomino  from  Acampo  

\       ° 

0 

puncheon  No.  12 

XI. 

Palomino  from  Acampo  

32  oz. 

plus 

puncheon  No.  13 

XII. 

Palomino  from  Acampo  

32  oz. 

plus 

puncheon  No.  14 

A  series  of  8  puncheons  was  filled  with  the  free  run  must  of  a  car- 
load of  Green  Hungarian  grapes  from  Acampo.  The  grapes  were  in 
rather  poor  condition,  for,  being  very  juicy  and  thin  skinned,  many 
were  broken,  and  the  natural  micro-organisms  were  present  in  large 
numbers. 

Three  puncheons  (fermentations  I,  II,  and  III)  were  filled  with  the 
must  and  allowed  to  ferment  spontaneously  without  any  addition.  One 
puncheon  (IV)  was  sulfited  at  the  rate  of  12  ounces  of  metabisulfite 
to  200  gallons  and  allowed  to  ferment  without  a  starter.  The  other 
four  puncheons  (V,  VI,  VII,  and  VIII)  were  sulfited  in  the  same  way, 
but  after  settling  for  forty-eight  hours,  were  drawn  off  the  sediment 
into  clean  puncheons  and  started  with  pure  yeast.  A  larger  lot  of  the 
same  must  was  defecated  in  the  same  way  in  a  1,700  gallon  vat  and, 
after  settling,  drawn  off  into  a  1,500  gallon  cask  and  fermented  with 
pure  yeast. 


104 


UNIVERSITY  OF  CALIFORNIA — EXPERIMENT  STATION. 


The  following  partial  records  show  the  progress  of  some  of  the  fer- 
mentations. 

TABLE  No.  49. 
Green  Hungarian.     No  sulfite.     No  pure  yeast. 


Fermentation  I. 


Balling. 


Temp. 


Fermentation  II.  • 


Fermentation  IH. 


Hours. 


Balling. 


Temp. 


Hours. 


Balling. 


Temp. 


0 

44 
54 


20 
17 
10 

3.7 

0 


73.5 
82.5 
79.5 


0 

24 
34 
46 
50 


18 
16 
10 
7.5 


64.5 


74.5 
75.0 


0 

48 
52 

55 

72 


20 

10 
7.8 
5.3 
0 


70 
72.5 


78 


♦Record  incomplete. 


TABLE  No.   50. 
Green  Hungarian. 


Fermentation 

IV.     Sulfite  12  ounces. 

Fermentation  V.     Sulfite  12  ounces. 

Fermentation  IX.     1500-gallon  cask. 

No  pure  yeast 

Pure  yeast. 

Sulfite  12 

ounces.     Pure  yeast. 

Hours. 

Balling. 

Temp. 

Hours. 

Balling. 

Temp. 

Hours. 

Balling. 

Temp. 

0 

20.0 
20.0 

0  

18 

19.6 

19.2 

59.0 
61.0 

0 

40  

21 
20 

61.5 

72 

90 

17.0 

62.5 

28  

18.0 

63.5 

136  

19.7 

112 

16.0 

61.0 

42 

16.0 

65.5 

160  

17.0 

68.0 

140  

15.2 

63.5 

50 

14.0 

66.5 

170  

15.5 

73.5 

146  

13.0 

65.0 

64 

9.8 

72.5 

194 

10.0 

80.5 

160 

7.5 

71.5 

69 

7.5 

78.0 

199 

6.5 

83.0 

165  

5.2 

74.5 

88  

4.0 

77.0 

218 

4.0 

86.0 

170 

4.5 

76.0 

93  

2.5 

77.0 

228  

3.0 

88.0 

189 

0 

79.0 

98  

2.0 

76.0 

10  days. 

2.5 

81.0* 

112  

.5 

73.5 

21  days. 

2.0 

72.5t 

117 

0 

73.5 

22  days. 

1.5 

24  days. 

1.0 

26  days. 

.5 

31  dayS- 

0 

*  Pumped  over  to  aerate. 
tNew  yeast  added. 


TABLE  No.  51. 

Green  Hungarian.     Sulfite,  12  ounces,  and  pure  yeast. 


Fermentation  VI. 


Fermentation  VII. 


Fermentation  VIII. 


Balling. 


0 

21 

45 

69 

93 

117 

141 

151 

165 

175 

189 

199 

223 


18.6 

18.4 

18.4 

18.2 

18.0 

17.5 

15.0 

12.5 

9.0 

6.2 

3.7 

2.0 

0 


Temp. 


61.5 
59.0 
62.0 
62.0 
64.5 
66.0 
66.0 
70.0 
73.5 
77.0 
74.5 
74.5 
75 


0 

15 

25 

49 

63 

67 

71 

85 

95 

109 

119 

133 

157 

181 

205 

215 


Balling. 


19.1 

19.1 

18.5 

18.0 

17.0 

16.5 

16.0 

14.0 

12.0 

10.0 

7.5 

5.5 

3.0 

2.0 

1.0 

0 


Temp. 


62.5 
59.0 
61.5 
62.5 
62.5 
69.0 
68.0 
68.0 
69.0 
68.0 
70.0 
70.5 
71.5 
70.0 
69.0 
71.5 


0 

16 

40 

64 

74 

88 

98 

112 

122 

136 

160 

184 

208 

232 


Balling. 


18.1 
17.9 
17.5 

17.5 

16.0 

15.0 

13.25 

10.5 

10.0 

6.0 

3.5 

2.0 

1.0 

0 


Temp. 


62.5 
61.5 
61.5 
62.5 
68.0 
68.0 
68.0 
70.0 


70.0 
73.5 
72.5 
70.5 
70.0 


Bulletin  230] 


ENOLOGICAL    INVESTIGATIONS. 


105 


The  curves  of  several  typical  examples  of  these  fermentations  are 
shown  in  diagram  13. 


SULF  Tf  N( )  PURt  YEAST 

W  sIjlfi  tc.no  =>URE  VERS 


= 


LFITtflMDFVfrEY&ST 
LFITERN&PURE  YER5T 


OOa?LV/97:   SULFITE 
WO  WREY£/9§T 


'igh&tfO  XG+  288  && 


Diagram  13. 
Fermentation  Curves  of  Green  Hungarian. 

These  show  a  notable  lengthening  of  the  fermentation  in  the  casks 
where  sulfite  was  used.  This  is  in  strong  contrast  with  the  results 
of  the  red  wine  fermentations  where  it  was  found  that  the  slight 
retarding  influence  of  the  S02  was  much  exceeded  by  the  acceleration 
due  to  the  use  of  a  starter. 

In  No.  I,  in  which  fermentation  was  allowed  to  start  spontaneously 
and  without  S02,  the  Balling  per  cent  had  fallen  to  0  in  less  than  3 
days.  This  showed  the  presence  of  a  large  amount  of  natural  yeast 
due  to  the  bad  condition  of  the  grapes.  In  No.  IV,  which  received  an 
addition  of  sulfite,  the  start  of  fermentation  was  delayed  for  several 
days.  The  actual  tumultuous  fermentation,  however,  was  almost  as 
rapid  as  that  of  No.  I,  but  the  total  time  needed  before  reaching  0 
Balling  was  over  8  days.  The  irregularity  of  the  curve  of  this  fermen- 
tation is  unaccounted  for,  but  may  have  been  due  to  the  presence  of  a 
mixture  of  fermentative  organisms  having  different  degrees  of  resistance 
to  S02  and  to  alcohol. 

Nos.  V,  VII,  and  IX  were  treated  with  sulfite,  defecated,  drawn  off 
the  sediment  and  started  with  pure  yeast.  The  regularity  of  Nos.  V 
and  VII  is  in  marked  contrast  with  No.  IV  and  is  doubtless  due  to  the 
uniformity  of  the  yeast.  The  retarding  influence  of  the  S02  is  very 
marked  in  No.  VII  but  much  less  so  in  No.  V.  This  seems  to  be  due  to 
the  fact  that  in  No.  VII  the  yeast  was  added  49  hours  after  the  sulfite 
and  in  No.  V  68  hours.  The  earlier  addition  of  the  yeast  exposed  it  to 
larger  amounts  of  free  S02  and  thus  weakened  its  fermenting  power. 
No  ill  effect  resulted  from  this  slackening  of  the  fermentation  as  both 


106  UNIVERSITY  OF   CALIFORNIA EXPERIMENT  STATION. 

musts  became  perfectly  dry  without  interruption.  The  slower  fermen- 
tation of  No.  VII  in  fact  resulted  in  a  lower  maximum  temperature 
than  in  No.  II,  which  is  an  advantage.  Sulfurous  acid  thus  seems  to 
be  capable  of  controlling  the  temperature  to  some  extent  in  white  wine 
fermentation,  contrary  to  the  results  shown  in  the  red  wine  fermenta- 
tions where  no  appreciable  effect  of  this  kind  was  noted. 

In  No.  IX  the  yeast  was  added  only  30  hours  after  the  sulfite  and 
we  have  a  correspondingly  greater  delay  in  the  fermentation.  The  delay 
in  this  case  did  not  result  in  a  lower  maximum  temperature  on  account 
of  the  large  volume  of  fermenting  must  (1500  gallons)  which  retarded 
the  escape  of  heat.  Fermentation  in  No.  IX  did  not  start  perceptibly 
until  over  5  days  after  the  addition  of  the  yeast,  while  in  the  sulfited 
musts  in  puncheons  it  was  noticeable  in  from  24  to  48  hours.  The 
fermentation  after  it  started  was  rapid,  owing  to  the  conservation  of 
the  heat.  It  became  very  slow,  however,  by  the  ninth  day  while  the 
saccharometer  still  showed  3  per  cent  Balling.  By  the  tenth  day  the 
sugar  had  fallen  only  2.5  per  cent  Balling.  A  thorough  aeration  on 
this  day  stimulated  the  fermentation  slightly  but  on  the  twenty-first 
day  2  per  cent  Balling  still  remained ;  only  .5  per  cent  Balling  having 
been  eliminated  in  8  days. 

It  was  evident  that  the  yeast  was  weak  so  that  on  this  day  a  new 
addition  of  pure  yeast  was  made.  The  fermentation  revived  and  one 
and  a  half  per  cent  of  sugar  disappeared  in  5  days.  The  wine  finally 
reached  0  per  cent  Balling  on  the  thirty-first  day. 

The  length  of  this  fermentation  was  due  in  the  first  place  to  the  use 
of  more  sulfurous  acid  than  was  necessary  for  the  grapes,  which  only 
had  21  per  cent  Balling  and  for  the  low  temperature  of  the  must  which 
was  61.5°  F.  at  the  start.  In  No.  V,  where  sufficient  time  was  allowed 
for  the  S02  to  combine  before  adding  the  yeast,  no  harm  was  done. 
In  No.  VII,  where  the  must  was  placed  in  a  puncheon,  there  was  suf- 
ficient aeration  to  keep  the  fermentation  going.  In  No.  IX,  however, 
the  yeast  was  added  too  soon  and  the  large  size  of  the  fermenting  cask 
diminished  the  amount  of  aeration,  resulting  in  a  deficiency  of  oxygen 
for  the  yeast  and  a  slower  disappearance  of  the  free  S02. 

All  these  cellar  tests  corroborate  the  conclusion  drawn  from  the 
laboratory  tests  reported  in  Part  II  that  the  effect  of  sulfurous  acid  is 
due  almost  entirely  to  the  free  S02,  and  that  the  S02  remains  free 
longer  and  in  larger  amounts  in  must  alone  than  in  the  whole  mass  of 
crushed  grapes.  Contrary  to  the  usual  practice,  therefore,  more  sul- 
furous acid  is  needed  and  should  be  used  in  red  wine  fermentations 
than  in  white. 

In  fermentation  X,  XI,  and  XII  the  effect  of  very  large  doses  of  S02 
on  excessively  moldy  grapes  was  tried.     The  results  were  not  favor- 


Bulletin  230] 


ENOLOGICAL    INVESTIGATIONS. 


107 


able.  All  the  good  effects  of  sulfurous  acid  were  obtained  with  small 
doses,  and  large  doses  simply  delay  the  fermentation  without  any  corre- 
sponding advantage  and  very  large  doses  injure  the  flavor  of  the  wine. 

In  a  general  way,  the  conclusion  is  that  for  the  conditions  under 
which  our  tests  were  made,  for  grapes  of  moderate  sugar  contents  and 
in  cool  weather  about  8  ounces  to  12  ounces  of  potassium  metabisulfite 
per  ton  give  the  best  results  in  the  fermentation  of  red  wine,  and  about 
half  of  this  amount  is  all  that  is  needed  in  the  defecation  and  fermen- 
tation of  white  wine. 

These  comparatively  small  amounts  are  sufficient  to  paralyze  immedi- 
ately all  the  micro-organisms  present,  which  is  the  main  object,  and 
then  quickly  change  to  the  combined  form  so  that  they  do  not  interfere 
with  the  work  of  the  yeast  which  is  added  later.  Where  moderation  of 
the  rate  and  temperature  of  fermentation  are  desirable  they  should  be 
obtained  by  cooling  methods  and  not  by  the  addition  of  excessively 
large  doses  of  S02. 

(d)  Quality  of  the  Wines. — In  estimating  the  influence  of  the  char- 
acter of  the  fermentation  on  the  quality  of  the  wine,  we  must  take 
into  consideration  the  data  supplied  by  tasting,  chemical  analysis  and 
microscopical  examination.  These  are  all  necessary  to  determine  how 
well  the  yeast  has  done  its  work,  how  well  the  work  of  injurious  micro- 
organisms has  been  controlled  and  what  the  probable  future  develop- 
ment of  the  wine  will  be. 

1.  sugar.  Owing  to  the  coolness  of  the  season  there  was  very  little 
trouble  in  fermenting  the  wines  down  to  dryness.  What  little  difficul- 
ties were  encountered  show  clearly  the  utility  of  the  proper  use  of 
sulfurous  acid  in  this  respect.  Wine  in  five  of  the  storage  casks  showed 
appreciable  amounts  of  unfermented  sugar  when  analyzed  on  Feb- 
ruary 3,  1912. 

TABLE  No.  52. 
Wines   showing  unfermented  sugar. 


Wines. 

Sugar,  per  cent. 

Vol.  acid. 

Dec.  16. 

Feb.  3. 

April  10. 

I,  II.    Zinfandel,  no  sulfite,  no  yeast 

.63 

.65 

.34 
1.60 

.31 
1.19 

.3 

.140 

X,  XI.     Zinfandel,  sulfite  12  ounces,  plus  yeast 

.078 

XV.    Barbera,  sulfite  5  ounces,  no  yeast 

1.60 

.075 

IX.    Green    Hungarian,    sulfite   12    ounces,    plus 
yeast 

.074 

XI.  Palomino,  sulfite  36  ounces,  plus  yeast 

.120 

The  Zinfandel  of  fermentations  I  and  II  which  was  fermented  in  the 
usual  manner  without  sulfite  or  pure  yeast  showed  .63  per  cent  of 


108  UNIVERSITY  OF  CALIFORNIA — EXPERIMENT  STATION. 

unfermented  sugar.  This  would  not  have  been  serious  if  the  wine  had 
been  sound,  but  the  volatile  acid  had  increased  to  .14  per  cent  and  a 
microscopical  examination  showed  very  large  numbers  of  long  rod- 
shaped  bacteria.  Such  a  wine  as  this,  if  left  to  itself,  is  sure  to  spoil 
completely  during  the  first  warm  weather  of  spring. 

The  Zinfandel  of  fermentations  X  and  XI  which  had  been  sulfited 
and  fermented  with  pure  yeast,  showed  .34  per  cent  of  unfermented 
sugar.  The  volatile  acid,  however,  was  normal  at  .078  per  cent  and 
the  microscope  showed  yeast  cells  of  uniform  appearance  with  only  an 
occasional  bacterial  rod.  Such  a  wine  can  be  safely  handled  by  the 
ordinary  cellar  methods  and  will  ferment  prefectly  dry  during  the 
spring. 

The  Barbera,  fermentation  XV,  which  was  sulfited  lightly,  but 
allowed  to  ferment  with  its  own  yeast  showed  1.6  per  cent  of  sugar  on 
February  3d.  The  volatile  acid  was  normal.  The  microscope  revealed 
many  yeast  cells  of  various  forms — ellipsoideus,  pastorianus,  and  apic- 
ulatus  types  and  a  few  bacteria.  By  April  10th  the  sugar  had  fallen 
to  .7  per  cent  and  there  was  every  indication  that  the  wine  would  fer- 
ment to  dryness  and  remain  sound. 

The  Green  Hungarian,  white  fermentation  IX,  in  1,500  gallon  cask, 
had  been  defecated  with  12  ounces  of  metabisulfite  and  fermented  with 
pure  yeast.  The  fermentation  had  been  very  long  and  on  February  3d 
the  wine  still  showed  .3  per  cent  of  unfermented  sugar,  but  the  volatile 
acid  was  quite  normal,  .074  per  cent.  The  microscope  showed  the  sedi- 
ment to  be  almost  entirely  composd  of  yeast  cells,  only  a  very  few 
bacteria  being  found.  This  wine  was  cloudy  and  inferior  to  that  fer- 
mented in  puncheons  but  will  undoubtedly  finally  become  dry  and  clear 
and  is  in  no  danger  of  spoiling. 

The  Palomino,  white  fermentation  XI,  was  made  from  extremely 
defective  grapes,  crushed,  moldy  and  smelling  of  vinegar.  The  yeast 
was  evidently  injured  by  the  large  quantity  of  sulfite  used,  and  the 
sulfited  wine  still  showed  1.19  per  cent  of  sugar  when  a  puncheon  of 
the  same  must  fermented  without  sulfite  was  practically  dry.  The 
sediment  showed  an  abundance  of  yeast  and  very  few  bacteria,  so  that 
there  is  no  doubt  that  the  wine  will  complete  its  fermentation  in 
time.  The  comparatively  high  volatile  acidity,  .12  per  cent,  is  probably 
caused  by  abnormal  work  of  the  yeast  due  to  its  weakened  condition. 
This  high  volatile  acidity  does  not  indicate  any  danger  of  spoiling  as  it 
would  if  caused  by  bacteria,  and  the  wine  is  in  no  danger  of  becoming 
unsound.  A  much  smaller  amount  of  sulfite,  however,  would  undoubt- 
edly have  given  better  results  without  causing  the  "sulfur"  taste  which 
the  wine  still  shows. 


Bulletin  230] 


ENOLOGICAL    INVESTIGATIONS. 


109 


2.  volatile  acidity.  In  Tables  53  and  54  are  given  the  various 
determinations  of  the  volatile  acidity  of  the  wines  made  at  intervals 
from  a  few  days  after  the  completion  of  fermentation  until  six  months 
after. 

TABLE  No.  53. 
Progress  of  volatile  acidity. 


Red  Wines. 


Volatile  acid,  per  cent. 


Oct.  16.       Oct.  18.       Nov.  14.     Dec.  16.       Feb.  3.    !  April  10 


I,    II.    Zinfandel;    no    sulfite,    no 
yeast    

III.  Zinfandel;  sulfite  5  ounces__. 

IV,  V.    Zinfandel;  sulfite  12  ounces 
plus  yeast 

VI.  Petite   Sirah;   no  sulfite,  no 
yeast    

VII,  VIII.    Petite  Sirah;  sulfite  10 
ounces  plus  yeast 

IX.  Zinfandel;  no  sulfite,  no  yeast* 

X,  XI.    Zinfandel;  sulfite  12  ounces 
plus  yeast  

XII,    XIII.      Bouschet;    sulfite    8 

ounces  plus  yeast  

XV.    Barbera;  sulfite  5  ounces 


.073 


.076 


.055 
.064 


.105 
.096 


.076 
.080 


.112 
.092 

.082 

.084 

.076 
.061 


.115 


.082 


.078 
.082 

.082 

.074 
.061 


.140 
.089 

.080 

.077 

.078 
.079 

.078 

.070 
.059 


.114 

.073 

.081 

.078 

.096 
.075 


*  This  wine 
gallon  casks. 


was  stored  in  180-gallon  puncheons.     All  the  others  in  1500-  to  3500- 

TABLE  No.  54. 
Progress  of  volatile  acidity. 


White  Wines. 


Volatile  acid,  per  cent. 


Nov.  6.      Dec.  16.       Feb.  3.      April  10 


I. 
III. 
IV. 


VII. 

VIII. 

IX. 

X. 

XI. 

XII. 


Green  Hungarian;  no  sulfite,  no  yeast 

Green  Hungarian;  no  sulfite,  no  yeast 

Green   Hungarian;    sulfite   12   ounces,   no 

yeast  

Green  Hungarian;   sulfite  12  ounces  plus 

yeast   

Green  Hungarian;   sulfite  12  ounces  plus 

yeast   

Green  Hungarian;   sulfite  12  ounces  plus 

yeast   ___ 

Green  Hungarian;  sulfite  12  ounces  plus 

yeast*   

Palomino;  no  sulfite,  no  yeast 

Palomino;  sulfite  36  ounces  plus  yeast 

Palomino;  sulfite  36  ounces  plus  yeast 


.064 
.070 

.048 

.064 

.065 

.061 

.061 


.073 

.070 

.079 

.066 

.064 

.066 
.068 
.100 
.080 


.065 
.071 

.065 

.076 

.075 

.068 

.067 
.065 
.100 
.090 


.072 


.076 

.065 

.066 

.074 
.065 
.120 
.102 


*This  wine  was  stored  in  a  1500-gallon  cask.  All  the  others  in  180-gallon 
puncheons. 

As  shown  by  Table  53  the  volatile  acidity  in  all  the  sulfited  red  wines 
was  normal,  showing  a  clean  fermentation  and  absence  of  bacterial 
action.  In  one  wine,  Zinfandel  I  and  II,  fermented  in  the  ordinary 
way  without  sulfites,  the  volatile  acidity  commenced  to  rise  almost 
immediately  after  the  end  of  the  tumultuous  fermentation  and  con- 
tinued so  long  as  the  observations  extended.  This  is  a  case  of  a 
" stuck' '  wine  and  shows  the  ordinary  course  of  a  young  wine  contain- 
ing a  remnant  of  unfermented  sugar  and  large  quantities  of  bacteria. 


110  UNIVERSITY  OF   CALIFORNIA — EXPERIMENT  STATION. 

In  the  Petite  Sirah  No.  VI,  also  an  ordinary  fermentation,  the  vola- 
tile acidity  remained  normal  for  about  four  months  following  the  vin- 
tage. During  the  first  warm  weather  of  spring,  however,  the  volatile 
acid  commenced  to  rise  and  by  April  10th  it  was  60  per  cent  higher 
than  that  of  Petite  Sirah  VII  and  VIII  fermented  with  sulfite  and 
pure  yeast.  In  this  case  the  ordinary  fermentation  and  that  in  which 
sulfite  was  used  were  successfully  brought  to  0  Balling  and  the  wines 
were  both  practically  dry  when  analyzed.  The  sulfited  wine  showed, 
under  the  microscope,  a  clean  sediment  with  very  few  bacteria,  while 
the  unsulfited  showed  large  numbers  of  bacteria  almost  from  the  begin- 
ning. In  April  these  bacteria  were  very  numerous.  They  consisted  of 
long  and  short  rods  and  were  not  vinegar  bacteria.  This  experiment 
shows  that  even  the  completion  of  the  fermentation  to  apparent  dryness 
does  not  always  prevent  the  increase  of  volatile  acid  in  unsulfited 
wines. 

The  remaining  unsulfited  wine,  Zinfandel  No.  IX  shows  a  perfectly 
normal  volatile  acidity  up  to  April  10th,  being  in  this  respect  practi- 
cally identical  with  the  sulfited  Zinfandel  Nos.  X,  XI  made  from  the 
same  grapes.  When  analyzed  on  February  3d  it  showed  only  .21  per 
cent  of  unfermented  sugar  while  the  sulfited  wine  showed  .34  per 
cent  at  the  same  date.  A  microscopical  examination  of  the  sediment 
showed  very  few  bacteria  in  the  sulfited  wine  but  a  larger  number  in 
the  unsulfited. 

Both  wines  were  prefectly  sound  and  if  handled  properly  were  in  no 
danger  of  deteriorating.  That  this  wine  No.  IX  kept  better  than  the 
others  fermented  in  the  same  way  without  sulfiting  seems  to  be  due  to 
the  fact  that  it  was  stored  in  puncheons  immediately  after  fermen- 
tation. This  view  is  corroborated  by  the  fact  that  there  was  no  abnor- 
mal increase  of  the  volatile  acid  in  any  of  the  Green  Hungarian  wines 
stored  in  puncheons  whether  sulfite  was  used  or  not.  The  increase  of 
volatile  acidity  after  fermentation  is  due  to  anaerobic  bacteria  which 
grow  the  more  vigorously  the  better  they  are  protected  from  oxygen. 
In  a  puncheon,  the  aeration  through  the  pores  of  the  wood  is  much 
greater  than  in  a  large  vat.  In  a  puncheon  of  180  gallons  the  ratio  of 
the  surface  to  the  volume  is  about  4J  times  greater  than  in  a  cask  of 
1500  gallons.  Supposing  the  wood  of  both  casks  to  be  of  the  same 
kind  and  thickness,  therefore,  the  wine  in  a  puncheon  is  aerated  by  the 
slow  penetration  of  air  through  the  staves  and  head  4J  times  as  much  as 
the  wine  in  a  1500  gallon  cask.  This  is  sufficient  to  account  for  the 
quicker  finish  of  the  fermentation  of  wines  stored  in  small  casks,  the 
smaller  growth  of  anaerobic  bacteria  and  the  better  development  of  the 
wine.  The  use  of  sulfurous  acid  prevents  the  excessive  growth  of  bac- 
teria even  in  the  large  casks,  but  it  does  not  overcome  the  slowness  of 
the  after  fermentation  or  of  the  development  of  the  wine. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


Ill 


3.  Fixed  Acidity.  The  influence  of  sulfite  and  pure  yeast  on  the 
fixed  acidity  is  masked  in  the  red  wines  by  unavoidable  variations  in 
the  grapes  and  by  the  difficulty  of  obtaining  a  representative  sample 
before  fermentation.  The  errors  thus  introduced  are  likely  to  neutral- 
ize each  other  to  some  extent  in  the  means  of  all  the  wines.  The 
figures  representing  the  average  acidity  found  in  the  red  grapes  and  in 
the  corresponding  red  wines  may  safely  be  taken  as  representing  the 
general  direction  of  the  influence  in  this  respect.  In  the  white  wines 
the  observational  errors  are  much  smaller.  The  white  grapes  were  all 
crushed  into  the  same  vat  and  the  must  from  this  vat  distributed  into 
the  various  vats  and  puncheons  in  which  the  experiments  were  made. 
The  variations  in  the  composition  of  the  raw  material  and  in  the 
samples  taken  for  analysis  are  much  less  in  this  case. 


TABLE  No.  55. 

Influence  of  sulfurous  acid  on  fixed  acidity. 


Alcohol. 

Fixed  acid. 

No  sulfite. 

Sulfite. 

Must. 

Wine. 

Must. 

Wine. 

RED    WINES. 

Zinfandel,  I,  II  ___ 

11.85 
12.60 
12.30 
11.65 
11.90 
13.40 
12.15 

1.06 

.51 

Zinfandel,  III;  5  ounces  sulfite 

1.00 

.97 

.38 

Zinfandel,  IV,  V;  12  ounces  sulfite 

.63 

Petite  Sirah,  VI  

.81 

.40 

Petite  Sirah,  VII,  VIII;  10  ounces 

.84 

.36 

Zinfandel,    IX   

.95 

.53 

Zinfandel,  X,  XI,  12  ounces 

.96 
.94 

.82 

Average  fixed  acidity  of  red  wines 

.94 

.64 
.64 

.48 

.45 
.43 

.55 

WHITE   WINES. 

Green  Hungarian,  I  

11.30 
11.30 
11.25 
11.20 
11.30 
11.30 
11.20 

Green  Hungarian,  III  

Green  Hungarian,  IV;  12  ounces  sulfite___ 

.64 
.64 
.64 
.64 

.64 

.64 

.57 

Green  Hungarian,  V;  12  ounces  sulfite 

.60 

Green  Hungarian,  VII;  12  ounces  sulfite__ 

.60 

Green  Hungarian,  VIII;  12  ounces  sulfite. 

.55 

Green  Hungarian,  IX;  12  ounces  sulfite  __ 

.51 

Average  fixed  acidity  of  white  wines 

.64 

.44 

.57 

This  table  indicates  that  in  the  red  wine  fermentations  where  no 
sulfite  was  used,  there  was  a  loss  of  49  per  cent  of  the  fixed  acidity. 
This  loss  was  reduced  to  41  per  cent  where  sulfites  were  used.  In  the 
white  wine  only  31  per  cent  of  the  fixed  acidity  was  lost  even  in  the 
ordinary  fermentations,  and  this  loss  was  reduced  by  the  use  of  sulfites 
to  11  per  cent. 

The  greater  loss  of  fixed  acidity  in  the  red  wines  is  accounted  for 
principally  by  the  more  advanced  stage  of  maturity  of  the  grapes  from 
which  they  were  made.     The  less  ripe  the  grapes  the  more  of  their 


112 


UNIVERSITY  OF   CALIFORNIA — EXPERIMENT  STATION. 


acidity  exists  in  the  form  of  free  tartaric  acid.  This  acid  is  soluble  in  all 
proportions  in  the  wine  and,  therefore,  is  found  in  toto  in  the  wine 
unless  destroyed  by  acid-consuming  organisms.  In  the  riper  grapes,  a 
greater  part  of  the  acidity  exists  in  the  form  of  bitartrate  of  potash 
which  is  less  soluble  in  the  wine  than  in  the  must.  A  certain  propor- 
tion of  this  acidity  is  therefore  lost  from  the  wine  by  the  precipitation 
of  cream  of  tartar.  This  loss  is  further  increased  by  the  production  of 
alcohol  in  which  the  bitartrate  is  insoluble.  The  more  alcoholic  the 
wine,  therefore,  the  less  bitartrate  can  remain  in  solution. 

The  increase  of  acidity  with  the  use  of  sulfurous  acid  is  due,  there- 
fore, principally  to  its  conservation  of  the  free  acid. 

4.  alcohol.  As  shown  by  Table  56  the  sulfited  red  wines  showed  a 
better  utilization  of  the  sugar.  The  ratio  of  the  alcohol  by  volume 
in  the  wine  to  the  original  Balling  °  of  the  must  being  on  the  average 
.55  for  the  sulfited  wines  and  .52  for  the  parallel  wines  fermented 
without  sulfites. 

TABLE  No.  56. 
Influence  of  S02  and  pure  yeast  on  alcohol  production  in  red  wines. 


No  sulfites. 

Sulfites. 

Fermentation. 

Balling  ° 
of  must. 

Alcohol  in 
wine. 

Ratio. 

Balling  ° 
of  must. 

Alcohol  in 
wine. 

Ratio. 

Zinfandel,   I,   II   „. 

24.8 

11.85 

.48 

Zinfandel,   III   

22.3 
22.5 

12.60 

12.40 

.57 

Zinfandel,  IV,  V 

.55 

Petite  Sirah,  VI 

22.5 

11.85 

.53 

Petite  Sirah,  VII,  VIII_ 

22.1 

11.85 

.54 

Zinfandel,  IX  

24.5 
23.8 
23.8 

13.40 
12.37 
13.57 

.55 
.52 
.57 

Means    -.1 

22.3 
22.3 

12.28 
12.49 

.55 

Theoretical  ratio  _ 

.56 

The  ratio  of  alcohol  to  Balling  °  should  be  higher  with  the  grapes 
containing  more  sugar,  as  the  proportion  of  non-sugar  extract  included 
in  the  Balling  °  is  less.  In  the  last  line  of  the  table  is  given  the  amount 
of  alcohol  and  the  ratio  which  should  have  been  obtained  according 
to  Salleron's  tables.  This  shows  an  average  loss  of  alcohol  in  the  unsul- 
fited  wines  of  1.2  per  cent  and  in  the  sulfited  of  only  .21  per  cent. 
These  losses  represent  partly,  unfermented  sugar,  and  partly,  sugar 
and  alcohol  wasted  by  wild  yeasts,  bacteria  and  high  temperatures. 

5.  Color  of  Red  Wines.  It  is  difficult  to  determine  exactly  the 
influence  exerted  by  the  S02  on  the  color  on  account  of  unavoidable 
variations  in  the  grapes  of  the  various  vats.  On  the  whole,  there  is  no 
doubt,  however,  that  it  was  favorable.  The  wines  from  the  sulfited 
vats  in  nearly  all  instances  were  of  better  tint  and  more  intense  colors 
than  those  from  the  corresponding  unsulfitcd  vats. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


113 


TABLE  No.  57. 
Influence  of  S02  and  pure  yeast  on  the  color  of  red  wines. 


Fermentation. 

No  sulfite. 

Sulfite. 

Zinfandel,  I,  II 

5VR 

44 

Zinfandel,  III  

4VR 
4VR 

35 

Zinfandel,  IV,  V  -                    

52 

Petite  Sirah,  VI __ 

2VR 

93 

Petite  Sirah,  VII,  VIII  _                                           

1VR 

121 

Zinfandel,  IX _  _  _ 

3VR 

35 

Zinfandel,  X,  XI .       

3VR 

66 

Means  

56 

69 

In  the  Zinfandel  of  the  first  series  the  sulfited  wines  show  a  slight 
improvement  of  tint  but  the  intensity  of  the  color  is  in  one  case  slightly 
less  and  in  the  other  slightly  more  than  in  the  unsulfited.  This  is 
really  a  favorable  showing,  as  the  unsulfited  grapes  were  riper  and 
therefore  more  deeply  colored.  In  the  other  series  the  sulfited  showed 
from  30  per  cent  to  80  per  cent  more  color  than  the  corresponding 
unsulfited  wines.  The  average  of  all  the  wines  showed  an  increase 
of  about  23  per  cent. 

6.  condition.  The  condition  of  a  young  wine  means  its  degree  of 
limpidity  or  the  amount  and  character  of  suspended  solid  matter  it 
contains.  A  wine  during  and  immediately  after  fermentation  is  said 
to  be  "murky  or  muddy."  After  the  main  part  of  the  yeast  and  other 
sediment  has  settled  it  is  said  to  be  "cloudy."  When  it  is  sufficiently 
freed  from  floating  matter  to  be  transparent  it  is  called  "clear."  The 
final  stage  of  limpidity  is  reached  when  the  most  careful  scrutiny 
fails  to  detect  any  visible  floating  particles.  The  wine  is  then  said  to 
be  "bright." 

These  various  stages  occupy  various  periods  according  to  the  soundness 
of  the  wine  and  the  methods  of  handling.  A  wine  will  lose  its  gross 
sediment  and  reach  the  "cloudy"  stage  within  a  few  days  after  the 
end  of  perceptible  fermentation.  The  length  of  time  during  which 
wines  remain  "cloudy"  will  vary  from  a  few  days  to  many  months. 
A  perfectly  sound  and  properly  fermented  wine  should  be  "clear" 
within  three  weeks  of  pressing  in  the  case  of  red  wine  and  within  six 
or  eight  weeks  in  the  case  of  white.  Clearing  is  delayed  by  unfer- 
mented  sugar  and  the  presence  of  many  bacteria.  It  is  hastened  and 
facilitated  by  suitable  aeration  and  temperature. 

The  rapid  clearing  of  the  wine  was  a  constant  effect  of  the  use  of 
sulfurous  acid  and  pure  yeast  owing  to  the  more  perfect  elimination 
of  the  sugar  and  the  prevention  of  bacterial  and  wild  yeast  action. 
7—230 


114 


UNIVERSITY  OF   CALIFORNIA EXPERIMENT   STATION. 


7.  sulfurous  acid  remaining  in  the  wine.  In  Table  58  is  shown 
the  amount  of  total  S02  found  in  the  wines  fermented  in  the  presence 
of  sulfites.  For  the  white  wines  the  amount  of  free  S02  found  is  also 
given.  The  determinations  were  made  at  the  beginning  of  February 
when  the  wines  were  about  four  months  old. 


TABLE  No.  58. 
Sulfurous  acid  remaining  in  the  wine. 


Red  wines. 


Sulfite  added 
per  ton. 


Equivalent 
S02  per  cent. 


Per  cent  of 

S02  found 

in  wine. 


Zinfandel,  III  _ „ 

Zinfandel,  IV,  V 

Petite  Sirah,  VII,  VIII 

Zinfandel,  XXI  

Alicante  Bouschet,  XII,  XIII 

Zinfandel,  XVI  

Miscellaneous    


5  oz. 
12  oz. 
10  oz. 
12  oz. 

8  oz. 

8  oz. 

8  oz. 


.0078 
.0188 
.0156 
.0188 
.0125 
.0125 
.0125 


.0040 
.0076 
.0076 
.0096 
.0108 
.0108 
.0096 


White  wines. 


Free  S02 

per  cent 

found  in 

wine. 


S02  per  cent 
added. 


Per  cent  of 
S02  found 


Green  Hungarian,  IV  _ 
Green  Hungarian,  V  __ 
Green  Hungarian,  VII 
Green  Hungarian,  VIII 
Green  Hungarian,  IX  _ 

Palomino,  XI  ___ 

Palomino,  XII 


.0013 
.0013 
.0012 
.0010 
.0013 
.0015 
.0015 


.0188 
.0188 
.0188 
.0188 
.0188 
.0500 
.0500 


.0154 
.0149 
.0154 
.0159 
.0151 
.0418 
.0340 


These  analyses  show  that  at  four  months  from  14  per  cent  to  60  per 
cent  of  the  total  sulfurous  acid  added  to  the  must  had  disappeared 
from  the  red  wine  and  from  16  per  cent  to  30  per  cent  from  the  white. 
There  is,  therefore,  no  danger  of  exceeding  the  legal  limit,  which  is 
sufficiently  high  for  all  the  legitimate  purposes  of  the  wine-maker. 
Even  in  the  Palomino,  where  from  3  to  4  times  as  much  sulfite  was 
used  as  was  necessary  or  desirable,  the  S02  in  one  puncheon  had 
already  fallen  below  the  legal  limit. 

The  amount  of  free  S02  was  very  low  in  all  the  wines— so  low  that 
in  the  red  wines  its  exact  determination  was  difficult. 


Bulletin  230] 


ENOLOGICAL   INVESTIGATIONS. 


115 


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116  UNIVERSITY  OF   CALIFORNIA — EXPERIMENT  STATION. 

V.    SUMMARY  AND  CONCLUSIONS. 

I.  Introduction. 

The  principal  modern  improvements  in  wine-making  are  the  intelli- 
gent use  of  sulfurous  acid,  pure  yeast  and  temperature  control. 

II.  Use  of  Sulfurous  Acid  in  Wine-Making. 

The  use  of  sulfurous  acid  in  wine-making  is  of  great  antiquity  but 
has  been  much  developed  and  perfected  within  the  last  twenty-five 
years. 

Approved  by  all  enological  experts. 

Present  legal  limitations  satisfactory  but  unnecessary  for  wine- 
making. 

Fumes  of  burning  sulfur  the  cheapest  source  of  S02  and  the  best  for 
disinfection  purposes. 

For  the  control  of  fermentation  this  source  is  uncertain  and  difficult 
to  regulate. 

From  1.5  pounds  to  2  pounds  of  sulfur  can  be  burned  in  a  1000 
gallon  cask. 

This  will  supply  about  .02  per  cent  of  S02  to  the  must  as  a  maximum. 

In  practice  usually  much  less  than  this  is  absorbed. 

Liquid  S02  appears  to  be  the  best  form  for  the  control  of  fermen- 
tation but  is  at  present  unavaliable  in  California. 

Solutions  of  S02  in  water  and  alcohol  are  unreliable. 

Potassium  metabisulfite  is  the  best  form  at  present  available. 

S02  disappears  less  rapidly  from  must  than  from  water. 

S02  combines  rapidly  with  certain  substances  in  the  must  into  which 
it  is  introduced. 

The  combination  of  S02  is  more  rapid  in  must  from  dried  grapes 
than  in  that  from  fresh. 

About  one  half  of  the  S02  disappears  during  the  fermentation  of 
red  wine. 

S02  is  more  stable  in  wine  than  in  must. 

The  increase  of  sulfates  in  the  wine  due  to  sulfiting  is  very  small. 

Very  ripe  grapes  require  more  S02  than  those  which  are  less  ripe  both 
because  they  are  more  liable  to  bacterial  attack  and  because  they  neu- 
tralize more  of  the  antiseptic  effect  of  the  S02. 

This  neutralization  of  the  antiseptic  effect  seems  to  be  due  to  neither 
excess  of  sugar  nor  to  deficiency  of  acidity. 

The  more  solid  matter  present  the  less  effective  the  S02.  Therefore, 
cloudy  must  requires  more  than  clear  and  the  crushed  grapes  in  red 
wine  fermentations  require  about  twice  as  much  as  the  must  in  white 
wine  fermentations. 

The  neutralizing  effect  of  must  is  increased  by  heating  to  120°  C. 
under  pressure. 


Bulletin  230]  ENOLOGICAL  INVESTIGATIONS.  117 

The  neutralization  of  the  antiseptic  power  of  the  S02  is  due  to  its 
combination  with  components  of  the  must. 

Free  S02  has  more  than  thirty  times  as  much  disinfecting  power 
as  combined  S02. 

Exposure  to  300  milligrams  per  liter  of  free  S02  will  kill  wine  yeast 
in  24  hours. 

About  50  milligrams  per  liter  of  free  S02  will  prevent  fermentation 
with  wine  yeast. 

Free  S02  is  about  sixty  times  as  effective  in  preventing  fermenta- 
tion as  combined  S02. 

Very  small  amounts  of  S02  (5  ounces  of  potassium  metabisulfite 
per  ton  in  most  cases)  are  sufficient  to  prevent  all  growths  of  molds  and 
wild  yeasts  and  to  insure  a  pure  fermentation  when  a  starter  of  wine 
yeast  is  used. 

100  milligrams  of  S02  per  liter  (6  ounces  of  potassium  metabisulfite 
per  ton)  eliminate  over  99.9  per  cent  of  the  active  cells  of  micro- 
organisms from  the  must. 

"Wine  yeast  does  not  become  inured  to  S02  but,  on  the  contrary, 
exposure  increases  its  sensibility.  The  reputed  training  of  yeast  to 
withstand  S02  is  a  fallacy. 

Wine  yeast  is  less  sensitive  to  S02  than  any  of  the  common  yeasts, 
molds  or  bacteria  occurring  in  grapes  and  wine. 

By  properly  timing  the  sulfiting  and  the  addition  of  the  starter  the 
full  effect  of  the  maximum  amount  of  free  S02.  is  exerted  on  the 
injurious  organisms  and  the  yeast  is  exposed  only  to  the  minimum 
amount  of  free  S02. 

III.     Utility  and  Methods  of  Application  of  Pure  Yeast  in  Wine-Making. 

Favorable  results  from  the  use  of  pure  yeast  in  wine-making  were 
obtained  by  the  station  nineteen  years  ago. 

Pure  yeast  has  been  used  regularly  with  success  for  many  years  in 
several  Californian  wineries. 

The  necessity  of  a  proper  selection  of  yeast  has  been  demonstrated. 

Champagne  and  Burgundy  yeasts  have  been  found  especially  suit- 
able to  Californian  conditions. 

Grapes  from  all  regions  investigated,  whatever  their  variety,  con- 
dition or  stage  of  ripeness  showed  large  numbers  of  mold  spores  and 
wild  yeasts.  The  wine  yeast  was  never  present  in  large  numbers  and 
was  usually  outnumbered  many  thousand  times  by  the  injurious  micro- 
organisms. 

Solid  cultures  of  pure  yeast  were  found  more  stable  and  reliable  than 
liquid  cultures. 

Direct  application  of  the  pure  yeast  received  from   a  laboratory 


118  UNIVERSITY  OF   CALIFORNIA — EXPERIMENT  STATION. 

involves  the  buying  and  transporting  of  large  amounts  and  is  too 
expensive. 

The  previous  preparation  of  a  starter  from  a  small  culture  received 
from  a  yeast  laboratory  is  preferable. 

With  a  little  practice  and  care  any  intelligent  wine-maker  can  pre- 
pare a  starter. 

A  starter  should  be  used  when  it  has  its  maximum  efficiency  which 
is  about  the  stage  at  which  the  Balling  °  has  been  reduced  one  half. 
The  efficiency  does  not  diminish  much  until  all  the  sugar  has  disap- 
peared. 

Expensive  and  complicated  yeast  propagators  are  unnecessary  in 
wineries. 

A  simple  and  cheap  apparatus  can  be  devised  suitable  for  wineries 
of  any  size  and  requiring  the  labor  of  only  one  man  in  the  largest 
winery. 

IV.     Tests  of  the  Use  of  S02  and  Pure  Yeast  In  a  Winery. 

The  use  of  S02  and  of  pure  yeast  witout  the  aid  of  cooling 
appliances  was  tested  in  a  small  winery. 

The  growth  of  molds  and  wild  yeasts  was  completely  prevented  by 
the  use  of  S02. 

Large  quantities  of  S02  in  white  wine  making,  even  with  grapes  in 
very  bad  condition,  were  found  unnecessary  and  inadvisable. 

The  yeast  apparatus  used  was  satisfactory  and  insured  practically 
pure  fermentations. 

S02  and  pure  yeast  can  be  used  to  insure  the  completion  of  fermen- 
tation and  to  prevent  " stuck"  wines  even  without  cooling  devices  if 
the  wine  is  handled  in  small  casks. 

In  large  casks  and  vats  where  the  end  fermentation  is  often  slow  the 
use  of  S02  much  decreased  the  danger  of  bacterial  deterioration. 

The  volatile  acidity  is  uniformly  lower  in  the  sulfited  wines. 

The  fixed  acidity  is  protected  by  the  use  of  S02  and  the  sulfited 
wines  show  a  higher  total  acidity  than  the  others. 

The  use  of  sulfites  gave  an  increase  in  the  alcohol  of  the  wines  of 
about  1  per  cent. 

The  color  of  the  sulfited  red  wines  was  improved  in  both  tint  and 
intensity. 

The  treated  wines  cleared  more  rapidly  and  showed  sounder  sedi- 
ments. 

The  amount  of  S02  remaining  in  the  wines  was  much  below  the  legal 
limitation  except  where  unnecessarily  large  amounts  were  used. 


STATION    PUBLICATIONS  AVAILABLE   FOR  DISTRIBUTION. 


REPORTS. 

1896.  Report  of  the  Viticultural  Work  during  the  seasons  1887-93,  with  data  regard- 

ing the  Vintages  of  1894-95. 

1897.  Resistant  Vines,    their   Selection,   Adoption,   and   Grafting.     Appendix   to   Viti- 

cultural Report  for  1896. 

1902.  Report  of  the  Agricultural  Experiment  Station  for  1898-1901. 

1903.  Report  of  the  Agricultural  Experiment  Station  for  1901-03. 

1904.  Twenty-second  Report  of  the  Agricultural  Experiment  Station  for  1903-04. 


BULLETINS. 


Reprint. 
No.   128. 

133. 
147. 
162. 
164. 
165. 
167. 
168. 
169. 

170. 

171. 

174. 
176. 

177. 

178. 
179. 

181. 
182. 


183. 
184. 


185. 

186, 
187. 


188. 
189. 


191. 
192. 


193. 


194. 


Endurance  of  Drought  in  Soils  of 
the  Arid  Regions. 

Nature,  Value,  and  Utilization  of 
Alkali  Lands,  and  Tolerance  of 
Alkali.  (Revised  and  Reprint, 
1905.) 

Tolerance  of  Alkali  by  Various 
Cultures. 

Culture  Work  of  the  Sub-sta- 
tions. 

Commercial  Fertilizers.  (Dec.  1, 
1904.) 

Poultry  Feeding  and  Proprietary 
Foods. 

Asparagus  and  Asparagus  Rust 
in  California. 

Manufacture  of  Dry  Wines  in 
Hot  Countries. 

Observations  on  Some  Vine  Dis- 
eases in  Sonoma.  County. 

Tolerance  of  the  Sugar  Beet  for 
Alkali. 

Studies  in  Grasshopper  Control. 

Commercial  Fertilizers.  (June 
30,    1905.) 

A  New  Wine-cooling  Machine. 

Sugar  Beets  in  the  San  Joaquin 
Valley. 

A  New  Method  of  Making  Dry 
Red  Wine. 

Mosquito  Control. 

Commercial  Fertilizers.  (June, 
1906.) 

The  Selection  of  Seed- Wheat. 

Analyses  of  Paris  Green  and 
Lead  Arsenic.  Proposed  In- 
secticide Law. 

The   California   Tussock-moth. 

Report  of  the  Plant  Pathologist 
to  July  1,  1906. 

Report  of  Progress  in  Cereal 
Investigations. 

The  Oidium  of  the  Vine. 

Commercial  Fertilizers.  (Janu- 
ary,  1907.) 

Lining  of  Ditches  and  Reservoirs 
to  Prevent  Seepage  and  Losses. 

Commercial  Fertilizers,  (June, 
1907.) 

California  Peach  Blight. 

Insects  Injurious  to  the  Vine  in 
California. 

The  Best  Wine  Grapes  for  Cali- 
fornia ;  Pruning  Young  Vines ; 
Pruning  the  Sultanina.     . 

Commercial  Fertilizers.  •  (Dec, 
1907.) 


No.   195.  The      California      Grape      Root- 
worm. 

197.  Grape     Culture     in     California; 

Improved  Methods  of  Wine- 
making  ;  Yeast  from  California 
Grapes. 

198.  The  Grape  Leaf- Hopper. 

199.  Bovine  Tuberculosis. 

200.  Gum  Diseases  of  Citrus  Trees  in 

California. 

201.  Commercial     Fertilizers.      (June, 

1908.) 

202.  Commercial  Fertilizers.     (Decem- 

ber,  1908.) 

203.  Report  of  the  Plant  Pathologist 

to  July  1,  1909. 

204.  The  Dairy  Cow's  Record  and  the 

Stable. 
205  Commercial  Fertilizers.     (Decem- 
ber,  1909.) 

206.  Commercial     Fertilizers.      (June, 

1910.) 

207.  The    Control    of    the    Argentine 

Ant. 

208.  The  Late  Blight  of  Celery. 

209.  The  Cream   Supply. 

210.  Imperial    Valley    Settlers'     Crop 

Manual. 

211.  How    to    Increase    the    Yield    of 

Wheat  in  California. 

212.  California  White  Oats. 

213.  The   Principles   of  Wine-making. 

214.  Citrus  Fruit  Insects. 

215.  The  Housefly   in   its   Relation   to 

Public  Health. 

216.  A  Progress  Report  upon  Soil  and 

Climatic  Factors  influencing  the 
Composition  of  Wheat. 

217.  Honey  Plants  of  California. 

218.  California  Plant  Diseases. 

219.  Report  of  Live  Stock  Conditions 

in  Imperial  County,  California. 

220.  Fumigation  Studies  No.  5  Dosage 

Tables. 

221.  Commercial     Fertilizers.       (Oct., 

1911.) 

222.  The  Red  or  Orange  Scale. 

223.  The  Black  Scale. 

224.  The     Production     of     the     Lima 

Bean. 

225.  Tolerance  of  Eucalyptus  for  Al- 

kali. 

226.  The  Purple  Scale. 

227.  Grape   Vinegar. 

228.  Pear     Thrips    and    Peach     Tree 

Borer. 

229.  Hog     Cholera     and     Proprietary 

Foods. 


CIRCULARS. 


No      1.     Texas  Fever. 

7.  Remedies  for  Insects. 
9.  Asparagus  Rust. 

10.  Reading  Course  in  Economic  En- 

tomology. 

11.  Fumigation  Practice. 

15.  Recent  Problems  in  Agriculture. 

What  a  University  Farm  is  For. 

29.  Preliminary    Announcement    Con- 

cerning Instruction  in  Practical 
Agriculture  upon  the  University 
Farm,  Davis,  Cal. 

30.  White  Fly  in  California. 

32.  White  Fly  Eradication. 

33.  Packing   Prunes    in    Cans.      Cane 

Sugar  vs.  Beet  Sugar. 

36.  Analysis  of  Fertilizers  for  Con- 
sumers. 

39.  Instruction  in  Practical  Agricul- 
ture at  the  University  Farm. 

46.  Suggestions  for  Garden  Work  in 
California  Schools. 

50.  Fumigation  Scheduling. 

52.  Information  for  Students  Con- 
cerning the  College  of  Agricul- 
ture. 


No.   55.  Farmers'     Institute    and    Univer- 
sity Extension  in  Agriculture. 

60.  Butter  Scoring  Contest,   1910. 

61.  University  Farm  School. 

62.  The  School  Garden  in  the  Course 

of  Study. 

63.  How    to    Make     an     Observation 

Bee  Hive. 

64.  Announcement    of   Farmers'  Short 

Courses  for  1911. 

65.  The  California  Insecticide  Law. 

66.  Insecticides  and  Insect  Control. 

67.  Development  of  Secondary  School 

Agriculture  in  California. 

68.  The  Prevention  of  Hog  Cholera. 

69.  The    Extermination    of    Morning- 

Glory. 

70.  Observations     on    the     Status    of 

Corn-growing  in  California. 

74.  Rice. 

75.  A  New  Leakage  Gauge. 

76.  Hot  Room  Callusing. 

77.  University  Farm  School. 

78.  Anouncement    of    Farmers'    Short 

Courses  for  1911. 

79.  List  of  Insecticide  Dealers. 


